Template directed split and mix synthesis of small molecule libraries

ABSTRACT

The invention combines the advantages of split and mix synthesis with the advantages of template directed synthesis. The method comprises the steps of: a) adding a linker molecule L to one or more reaction wells; b) adding a molecule fragment to each of said reaction wells; c) adding an oligonucleotide identifier to each of said reaction wells; d) subjecting said wells to conditions sufficient to allow said molecule fragments and said oligonucleotide identifiers to become attached to said linker molecule, or conditions sufficient for said molecule fragments to bind to other molecule fragments and sufficient for said oligonucleotide identifiers to bind to other oligonucleotide identifiers; e) combining the contents of said one or more reaction wells; and f) contacting the resulting bifunctional molecule(s) of step e) with one or more (oligonucleotide) templates each capable of hybridizing to at least one of the oligonucleotide identifiers added in step c).

FIELD OF INVENTION

The present invention relates to a method for synthesizing an encodedmolecule. Furthermore the invention pertains to a method for identifyinga molecule with desired characteristics and in certain aspects to alibrary of encoded molecules obtained by a method according to theinvention.

GENERAL BACKGROUND AND PRIOR ART

Methods are desired for increasing the efficiency of production andscreening of chemical libraries with the purpose of generation andisolation of new compounds that can be used for applications inmedicine, agriculture and other areas. Active molecules, for example foruse in medicine, have been identified by screening of natural materials(such as plant extracts) or chemical libraries of synthesised moleculesin assays that identify molecules with the desired properties. Theoutcome of such screens usually only is low affinity leads, i.e.molecules that are identified in the assay, (e.g. by binding to a targetmolecule or by another function), but which have insufficient affinityand specificity for the target. Further improvement of the leads istherefore required, which can be done empirically or by chemical design.In either case, the process of lead optimisation is time-consuming andexpensive and in many cases it does still not provide molecules withsufficient affinity and specificity for the target molecule to exert thedesired function with high efficiency and without unwanted reactions tooccur. Methods are required that increase the size of the compoundlibraries, in order to increase the diversity in the pool of moleculesthat is used for screening, and as the size of the libraries increaseimproved methods are required to identify the molecules that have thedesired properties in the screening assay.

DNA-encoding of compounds provides a means to perform more efficientscreens or selections where the isolated compound-DNA complexes can beidentified at the end by PCR-amplification, cloning and sequencing ofthe DNA portion (Lerner et al., EP0643778B1). In these DNA-encodedlibraries, each compound in the library is attached to a uniqueidentifier that “encodes” the chemical structure of the molecule towhich it is attached. This way, the structure of a molecule that isselected in the screening assay can easily be decoded by the attachedunique identifier. DNA-encoded libraries have also been generated bymeans of DNA-templating. In this approach, DNA templates direct thesynthesis of the encoded compounds (Walder et al., Proc. Natl. Acad.Sci. USA (1979), 76, 51-55; Bruick et al., Chemistry and Biology (1996),3: 49-56; Liu et al., WO02/074929A2; Pedersen et al., WO02/103008A2).Moreover, when these libraries are used in affinity selectionexperiments, the DNA of the recovered DNA-compound complexes can beamplified by PCR, and subsequently used in a new DNA-templated synthesisround, which directly amplifies the recovered compounds.

SUMMARY OF THE INVENTION

The present invention combines the non-templated technique of Lernerwith the templated technique of Walder and thereby provides an improvedmethod for the generation of oligonucleotide-encoded libraries.

In a primary aspect the invention pertains to a method for synthesizingan encoded molecule comprising the steps of:

-   -   a) Adding a linker molecule L to one or more reaction wells;    -   b) Adding a molecule fragment to each of said reaction wells;    -   c) Adding an oligonucleotide identifier to each of said reaction        wells;    -   d) Subjecting said wells to conditions sufficient to allow said        molecule fragments and said oligonucleotide identifiers to        become attached to said linker molecule, or conditions        sufficient for said molecule fragments to bind to other molecule        fragments and sufficient for said oligonucleotide identifiers to        bind to other oligonucleotide identifiers;    -   e) Combining the contents of said one or more reaction wells;    -   f) Optionally, distributing the combined product to one or more        new reaction wells;    -   g) Optionally, repeating steps b) to e) one or more times;

Contacting the resulting bifunctional molecule(s) of step e) or g) withone or more (oligonucleotide) templates each capable of hybridizing toat least one of the oligonucleotide identifiers added in step c);wherein

the linker molecule L contains at least one reactive group capable ofreacting with a reactive group in the molecule fragments and at leastone reactive group capable of reacting with a reactive group in theoligonucleotide;the molecule fragments each contain at least one reactive group capableof reacting with a reactive group in the linker molecule L or a reactivegroup in another molecule fragment, and the reactive groups of eachmolecule fragment may be the same or different;the oligonucleotide identifiers each contain at least one reactive groupcapable of reacting with a reactive group in the linker L or a reactivegroup in another oligonucleotide identifier, and the reactive groups ofeach oligonucleotide identifier may be the same or different;the oligonucleotide identifier added to each well in step c) identifiesthe molecule fragment added to the same well in step b);the steps a) to d) may be performed in any order;the steps b) to d) in step f) may also be performed in any order;the number of wells in steps a) and f) may be the same or different;the oligonucleotide template optionally is associated with a reactivegroup;

In another aspect, the invention relates to a method for identifying amolecule with desired characteristics, said method comprisingsynthesizing a library of encoded molecules by a method according to theinvention.

In a further aspect, the invention pertains to a library of encodedmolecules obtained or obtainable by a method according to the invention.

DEFINITIONS

As used herein, the term Bi-functional molecule means a bi-functionalmolecule consisting of an encoded molecule (e.g. a low molecular weightorganic molecule) and an oligonucleotide (e.g. a single- ordouble-stranded DNA molecule), where the oligonucleotide sequenceuniquely identifies the identity (structure) of the encoded molecule.The encoded molecule and the identifier are physically connected througha linker moiety. In certain embodiments, several oligonucleotides encodethe same encoded molecule, or several encoded molecules are encoded byone oligonucleotide (see below under “Library of bi-functionalmolecules”).

The bi-functional molecule can have one or more molecule fragmentsencoded by one or more oligonucleotide identifiers depending on thenumber of rounds of stage 1 split and mix synthesis used to generatedthe molecule.

Carrier molecule: Used interchangeably with carrier and bi-functionalcarrier molecule. A carrier molecule is a bi-functional molecule that isemployed in a Stage 2 templated synthesis, and may be generated by e.g.stage 1 synthesis. It thus consists of an encoded molecule (made up ofone or more molecule fragments) and an oligonucleotide identifier (madeup of one or more oligonucleotide identifiers) that uniquely identifies(encodes) the molecule fragment to which it is attached. Thebi-functional carrier molecule can have one or more molecule fragmentsencoded by one or more oligonucleotide identifiers (depending on thenumber of rounds of stage 1 split and mix synthesis used to generate thecarrier molecule).

Encoded molecule: The portion of the bi-functional molecule that isencoded by the oligonucleotide identifier of the bi-functional molecule.The encoded molecule is typically an organic molecule, typically ofrelatively low molecular weight compared to the oligonucleotideidentifier to which it is attached. The encoded molecule may be releasedfrom the identifier after its synthesis, to obtain the “free encodedmolecule”. The encoded molecule is typically attached to the identifierthrough a flexible linker.

Identifier: An oligonucleotide that encodes (specifies) the identity ofthe molecule fragment or encoded molecule to which it is attached. Forthe purpose of this invention, three kinds of identifiers are described:

Unit identifier, which is the oligonucleotide used in stage 1 synthesisto describe the identity of the molecule fragment that it becomesattached to, through the nascent bi-functional molecule, during asynthesis round in stage 1 synthesis.

Carrier identifier, which is the oligonucleotide component of a carriermolecule, i.e., the carrier identifier encodes the molecule fragment towhich it is attached.

Template identifier, also termed identifier template, encodes theencoded molecule attached to it after templated synthesis, or in caseswhere no templated synthesis is performed prior to the screening of thebi-functional molecules, encodes and identifies the encoded moleculeattached to it.

Library of bi-functional molecules: A library of bi-functional moleculesconsists of a number of bi-functional molecules, each of which consistsof an encoded molecule (e.g. low molecular weight organic molecules),attached to an identifier oligonucleotide (e.g. a single- ordouble-stranded DNA molecule), where the oligonucleotide sequenceuniquely identifies the identity (structure) of the encoded molecule towhich it is attached. In certain embodiments, several oligonucleotidesencode the same encoded molecule (i.e. several bi-functional moleculesin the library carry the same encoded molecule but differentoligonucleotide identifiers. In other embodiments, several differentencoded molecules are attached to the same oligonucleotide identifier.

Ligase enzyme: An enzyme that ligates together oligonucleotides. Ligaseenzymes may also be non-protein-based catalysts that mediate theligation of oligonucleotides, on single- or double-stranded form.

Ligation: A ligation reaction covalently links together molecules. It isprimarily used here to describe the ligation of two oligonucleotides toproduce one molecule, consisting of the two oligonucleotide sequences.

Linker L: The linker L is a molecule comprising a reactive group X,which is adapted for reaction with a molecule fragment, and a reactivegroup Z, which is adapted for ligation to an oligonucleotide fragment,and a linker Y, which connects X and Z.

Molecule fragment: A molecule fragment contains one or more reactivegroups that may react with reactive groups of other molecule fragments.

Molecular Entity: Used interchangeably with encoded molecule.

Nucleic acid: Used interchangeably with oligonucleotide.

Nucleic acid analog: Used interchangeably with oligonucleotide analogand unnatural oligonucleotide.

Nucleotide: Nucleotides as used herein refers to both natural andunnatural nucleotides. Oligonucleotides made up of nucleotides are thuscapable of sequence-specific hybridisation to natural oligonucleotidessuch as DNA and RNA. Nucleotides may differ from natural nucleotides byhaving a different phosphate moiety, sugar moiety, and/or base moiety.

Oligonucleotide: Oligonucleotides comprise a number of nucleotides asdefined above, i.e., oligonucleotides may comprise natural as well asunnatural nucleotides. Example oligonucleotides are thus DNA, RNA, PNA,morpholinos, and SNA, and may involve unnatural bases as well.

Reactive group: Reactive groups are capable of reacting with otherreactive groups to form a chemical bond. Reactive groups include —NH₂,—COON, —CHO, —OH, —NHR, —CSO₂OH, phenylchloride, —SH, —SS, and manyothers. Example pairs of reactive groups, and the resulting bondsformed, are shown in FIGS. 6 and 7. The reaction between two reactivegroups may be spontaneous under the conditions used, or can be catalyzedby enzymes, ribozymes or other organic or inorganic catalysts such asmetals. Furthermore, additional reagents may be added that reacts withthe reactive groups, in order to covalently link molecule fragments. Thelinkage between molecule fragments are thus typically of covalentcharacter. However, it may also be of non-covalent character. An exampleof such non-covalent bond between molecule fragments of an encodedmolecule is the bond formed when adding a molecule fragment comprising ametal-chelate complex (e.g., NTA-Zn++) to a nascent bi-functionalmolecule comprising an imidazole functionality.

Reactive units: Used herein interchangeably with reactive groups.

As used herein the term “well” defines a physical containment ofreagents, molecule fragments etc. in a localized space. A “well” thusinclude the well of a microtiter plate, any container, a spot of asolution on a glass plate, or other type of solid support (microarray),a reagent tube, a bead to which the reagents and molecules to be keptseparated are attached, and any other type of well that separatesdifferent compositions of reagents, molecule fragments etc. as desired.The separation does not have to be absolute, but should preferablyensure that the major components of a given well are the desiredcomponents. A nanocompartment where the molecule fragment to be attachedto the nascent bi-functional molecule is held in the vicinity of thereactive group of the bi-functional molecule, by hybridisation ofoligonucleotide strands, also is considered a “well”, since thehybridisation of the oligonucleotides keeps one reactive group (e.g., ofthe incoming molecule fragment) in localised space as seen from theother reactive group (e.g., of the bi-functional molecule). The complexof the bi-functional molecule and the incoming oligonucleotide-moleculefragment is therefore considered a nanocompartment and hence, under thisinvention, a “well”.

The terms ‘nucleic acid’, ‘nucleic acid molecule’ and ‘nucleic acidsequence’ as used herein refer to an oligomer or polymer of ribonucleicacid (RNA) or deoxyribonucleic acid (DNA) or mimics/mimetics thereof.This term includes molecules composed of naturally-occurringnucleobases, sugars and covalent internucleoside (backbone)phosphodiester bond linkages as well as molecules having non-naturallyoccurring nucleobases, sugars and covalent internucleoside (backbone)linkages which function similarly or combinations thereof. Such modifiedor substituted nucleic acids are often preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases and other enzymes, and are in thepresent context described by the terms “nucleic acid analogues” or“nucleic acid mimics”. Preferred examples of nucleic acidmimics/mimetics are peptide nucleic acid (PNA-), Locked Nucleic Acid(LNA-) xylo-LNA-, phosphorothioate-, 2′-methoxy-, 2′-methoxyethoxy-,morpholino- and phosphoramidate-containing molecules or the like.

The nucleic acid, nucleic acid molecule or nucleic acid sequence may,for instance, be composed entirely of deoxyribonucleotides, entirely ofribonucleotides, entirely of nucleic acid mimics or analogues orchimeric mixtures thereof. The monomers are typically linked byinternucleotide phosphodiester bond linkages. Nucleic acids typicallyrange in size from a few monomeric units, e.g., 5-40, when they arecommonly referred to as oligonucleotides, to several thousands ofmonomeric units. Whenever a nucleic acid or a nucleic acid sequence isrepresented, it will be understood that the nucleotides are in 5′ to 3′order from left to right and that “A” denotes deoxyadenosine, “C”denotes deoxycytidine, “G” denotes deoxyguanosine, and “T” denotesthymidine, unless otherwise noted.

In the present context ‘complementary sequence’ or ‘complement’ refersto nucleotide sequences which will anneal to a nucleic acid molecule ofthe invention under stringent conditions. The term “stringentconditions” refers to general conditions of high, weak or lowstringency. The term “stringency” is well known in the art and is usedin reference to the conditions (temperature, ionic strength and thepresence of other compounds such as organic solvents) under whichnucleic acid hybridisations are conducted. With “high stringency”conditions, nucleic acid base pairing will occur only between nucleicacid fragments that have a high frequency of complementary basesequences, as compared to conditions of “weak” or “low” stringency.

As an example, high stringency hybridisation conditions comprise (1) lowionic strength and high temperature for washing, such as 0.015 MNaCl/0.0015 M sodium citrate, pH 7.0 (0.1×SSC) with 0.1% sodium dodecylsulfate (SDS) at 50° C.; (2) hybridisation in 50% (vol/vol) formamidewith 5×Denhardt's solution (0.1% (wt/vol) highly purified bovine serumalbumin/0.1% (wt/vol) Ficoll/0.1% (wt/vol) polyvinylpyrrolidone), 50 mMsodium phosphate buffer at pH 6.5 and 5×SSC at 42° C.; or (3)hybridisation in 50% formamide, 5×SSC, 50 mM sodium phosphate (pH 6.8),0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon spermDNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C. with washesat 42° C. in 0.2×SSC and 0.1% SDS.

When referring to ‘identical sequences’ herein, again it is meantsequences having a certain degree of sequence identity. The sequencesmay thus be from 1-100%, such as at least 5%, at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 97%, at least 99% or 100% identical.

The term ‘sequence identity’ indicates a quantitative measure of thedegree of homology between two nucleic acid sequences of equal length.If the two sequences to be compared are not of equal length they must bealigned to give the best possible fit, allowing the insertion of gapsor, alternatively, truncation at the ends of the polypeptide sequencesor nucleotide sequences. The sequence identity can be calculated as

$\frac{\left( {N_{ref} - N_{dif}} \right)100}{N_{ref}},$

wherein N_(dif) is the total number of non-identical residues in the twosequences when aligned and wherein N_(ref) is the number of residues inone of the sequences. Hence, the DNA sequence AGTCAGTC will have asequence identity of 75% with the sequence AATCAATC (N_(dif)=2 andN_(ref)=8). A gap is counted as non-identity of the specific residue(s),i.e. the DNA sequence AGTGTC will have a sequence identity of 75% withthe DNA sequence AGTCAGTC (N_(dif)=2 and N_(ref)=8).

In all polypeptide or amino acid based embodiments of the invention thepercentage of sequence identity between one or more sequences is basedon alignment of the respective sequences as performed by clustalWsoftware (http:/www.ebi.ac.uk/clustalW/index.html) using the defaultsettings of the program. With respect to the nucleotide-basedembodiments of the invention, the percentage of sequence identitybetween one or more sequences is also based on alignments using theclustalW software with default settings. For nucleotide sequencealignments these settings are: Alignment=3Dfull, Gap Open 10.00, GapExt. 0.20, Gap separation Dist. 4, DNA weight matrix: identity (IUB).

In the present context, by ‘amplification reaction’ is meant a reactionthat produces one or more copies of a sequence of nucleic acids byrepeated extension of a probe or primer.

‘Extension’ may occur by virtue of polymerisation of individualnucleotide monomers, as in PCR, or it may occur by the addition ofprefabricated oligonucleotide segments, as in LCR, or by a combinationof these as in gap LCR or Repair Chain Reaction (RCR). Though notessential to the invention, ideally the extension reactions areperformed repeatedly and the extension products themselves may serve astemplates to produce an exponential generation of products.

‘T_(m)’ can be defined as the temperature at which 50% of a nucleic acidand its perfect complement are in duplex. The denaturation of doublestranded nucleic acids causes a shift in the absorbance of UV light at260 nm wavelength, an effect which can be assayed by determining theoptical density at 260 nm (OD₂₆₀). T_(m) is defined as the temperaturecorresponding to 50% denaturation, i.e. where the (OD₂₅₀) is midwaybetween the value expected for double stranded nucleic acids and thevalue expected for single stranded nucleic acids. The T_(m) of perfectlycomplementary duplexes can be calculated as follows:

T _(m)=81.5+16.6(log₁₀[Na⁺])+0.41(% GC)−500/length  DNA

T _(m)=79.8+18.5(log₁₀[Na⁺])+0.58(% GC)+11.8(% GC)²−820/length  RNA;RNA-DNA

T _(m)=2(no. of AT pairs)+4(no. of GC pairs)  Oligonucleotides

As used herein, ‘nucleic acid analogue’ Is understood to mean astructural analogue of DNA or RNA, designed to hybridise tocomplementary nucleic acid sequences (1). Through modification of theinternucleotide linkage(s), the sugar, and/or the nucleobase, nucleicacid analogues may attain any or all of the following desiredproperties: 1) optimised hybridisation specificity or affinity, 2)nuclease resistance, 3) chemical stability, 4) solubility, 5)membrane-permeability, and 6) ease or low costs of synthesis andpurification. Examples of nucleic acid analogues include, but are notlimited to, peptide nucleic acids (PNA), locked nucleic acids “LNA”,2′-O-methyl nucleic acids, 2′-fluoro nucleic acids, phosphorothioates,and metal phosphonates. Nucleic acid analogues are described in (2) and(3).

ABBREVIATIONS

L=Linker molecule.

R_(1(1-m))=Molecule fragments from repertoire 1. The repertoire used inthe corresponding round thus comprises m different molecule fragments.

R_(2(1-m))=Molecule fragments from repertoire 2. The repertoire used inthe corresponding round thus comprises m different molecule fragments.

O_(1(1-m))=Oligonucleotides coding for molecule fragments ofrepertoire 1. m different oligonucleotide sequences are thus used toencode the m different molecule fragments.

O_(1(1-m))=Oligonucleotides coding for molecule fragments of repertoire2. m different oligonucleotide sequences are thus used to encode the mdifferent molecule fragments.

R_(1, 1) thus is molecule fragment no. 1 of repertoire 1; R_(1, 2) ismolecule fragment no. 2 of repertoire 2; R_(2, 17) thus is moleculefragment no. 17 of repertoire 2; etc.

Oligo O_(1, 1) codes for molecule fragment R_(1, 1), oligo O_(1, 2)codes for molecule fragment R_(1, 2); oligo O_(2, m) codes for moleculefragment R_(2, m) etc.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method for combining the advantages of encodedmolecule fragments made by split and mix synthesis with the advantagesof template directed synthesis of molecules. The method provided in theinvention further has the advantage that molecules in the library afterselection in a screening assay can be easily identified and amplifiedfor use in subsequent screening procedures.

In outline, the invention combines methods for producing encodedmolecule fragments with methods for template directed synthesis ofmolecules. In the first step unique identifiers are attached todifferent molecule fragments, so that each different molecule fragmentis “encoded” by a unique identifier. In the next step, a second moleculefragment and thereafter a second identifier are attached. Like in thefirst step the identifier encodes the newly attached molecule fragment.This process can be continued until the desired number and diversity ofencoded molecules have been made. The resulting molecules are termedcarrier molecules, where a given carrier molecule contains organicmolecule fragments that are linked together, and where the linkedmolecule fragments are also linked to the identifiers that have alsobeen linked together.

These carrier molecules are hereafter brought together two-and-two bycomplementary binding of the identifier molecules to a template thatdirects which two carrier molecules are brought together. Thus, eachtemplate binds two carriers. In certain embodiments, the number ofcarriers that bind to the same template may be 3, 4, 5, 6, 7 or more.The proximity of the molecule fragments, once they are juxtaposed on thetemplate, allows for high fidelity chemical transfer reactions to occurin which one molecule fragment is transferred to the other and linked.In this way, the encoded molecule fragments produced by the firstmethods can be linked in all possible combinations to create very largelibraries of molecules. Since each molecule is defined by a template theidentity of the molecule, after it for example has been selected in ascreening assay, can easily be determined. Furthermore, the templates ofthe selected molecules can easily be amplified and used foramplification of the selected molecule fragments, which can then besubjected to further rounds of screening and selection. Each round ofscreening will thus enrich the pool of selected molecules with moleculeshaving the highest affinity or best function in the screening assay.

In a first aspect the invention pertains to a method for synthesizing anencoded molecule comprising the steps of:

-   -   a) Adding a linker molecule L to one or more reaction wells;    -   b) Adding a molecule fragment to each of said reaction wells;    -   c) Adding an oligonucleotide identifier to each of said reaction        wells;    -   d) Subjecting said wells to conditions sufficient to allow said        molecule fragments and said oligonucleotide identifiers to        become attached to said linker molecule, or conditions        sufficient for said molecule fragments to bind to other molecule        fragments and sufficient for said oligonucleotide identifiers to        bind to other oligonucleotide identifiers;    -   e) Combining the contents of said one or more reaction wells;    -   f) Optionally, distributing the combined product to one or more        new reaction wells;    -   g) Optionally, repeating steps b) to e) one or more times;    -   h) Contacting the resulting bifunctional molecule(s) of step e)        or g) with one or more (oligonucleotide) templates each capable        of hybridizing to at least one of the oligonucleotide        identifiers added in step c); wherein        the linker molecule L contains at least one reactive group        capable of reacting with a reactive group in the molecule        fragment and at least one reactive group capable of reacting        with a reactive group in the oligonucleotide;        the molecule fragments each contain at least one reactive group        capable of reacting with a reactive group in the linker molecule        L or a reactive group in another molecule fragment, and the        reactive groups of each molecule fragment may be the same or        different;        the oligonucleotide identifiers each contain at least one        reactive group capable of reacting with a reactive group in the        linker L or a reactive group in another oligonucleotide        identifier, and the reactive groups of each oligonucleotide        identifier may be the same or different;        the oligonucleotide identifier added to each well in step c)        identifies the molecule fragment added to the same well in step        b);        the steps a) to d) may be performed in any order;        the steps b) to d) in step f) may also be performed in any        order;        the number of wells in steps a) and f) may be the same or        different;        the oligonucleotide template optionally is associated with a        reactive group.

The process outlined above may be seen as a combination of a step 1synthesis and a step 2 synthesis, wherein the step 1 synthesis comprisesthe steps a) to g) and the step 2 synthesis is carried out in step h.

As mentioned, steps b) to e) may be repeated one or more times. It is tobe understood, however, that when step d) is performed for the firsttime the conditions must allow for the molecule fragments and saidoligonucleotide identifiers to become attached to said linker molecule.Accordingly, in the first round of step 1 synthesis, the conditions mustbe so that the molecule fragments do not only bind to other moleculefragments.

The present invention thus provides a method for the fast generation ofvery large libraries of DNA-encoded molecules in an amplifiable system.In the invention the synthesis of a library is carried out in two stagesby combination of two different methods. In stage 1 a repertoire ofdifferent bi-functional molecules are generated. These bi-functionalmolecules are called “bi-functional carrier molecules”, “carriermolecules” or simply “carriers”. Each bi-functional molecule consists ofa molecular entity and a unique identifier that codes for the molecularentity. In one embodiment of the invention the molecular entity is apolypeptide and the identifier encoding the polypeptide is a DNAoligonucleotide.

In stage 2, the carrier molecules produced in stage 1 are used in atemplated synthesis reaction, in order to create very large libraries ofmolecule fragments each attached to a unique identifier and each withthe ability of being amplified. In one embodiment the templates are DNAoligonucleotides that are complementary to the oligonucleotideidentifiers of the bi-functional carrier molecules produced in stage 1.

Stage 1:

In principle any method could be used for producing the bi-functionalcarrier molecules. In one embodiment this is done by split and mixsynthesis of the molecular entities and identifier molecules. In afurther embodiment this is done using the method in the exampledescribed below and illustrated in FIG. 1.

Example of Split and Mix Synthesis of Carriers:

The following example of split and mix synthesis of carriers is amodification of the method described by Lerner et al (Lerner, R. et al.(1993), European Patent Specification, “EP 0643778B1”).

The example is outlined in FIG. 1. In round 1, a linker molecule isfirst added to wells in a microtitre plate. Repertoires of differentamino acids (R, 1 through m) are hereafter added to the wells, one typeof amino acid per well (i.e., a specific amino acid to each well), andoperatively linked to the linker molecule. Other types of moleculefragments can be used as well, but in the present example amino acidsare used. Furthermore, a unique identifier (O, 1 through m), here a DNAoligonucleotide, is added to each well and operatively linked to thelinker molecule. Each well now contains a bi-functional molecule thatconsists of a linker molecule linked to an amino acid and an identifieroligonucleotide. Each well has a different amino acid and each aminoacid is linked, via the linker, to a unique DNA oligo. The sequence ofthe oligo encodes the type of amino acid added to that well. Theoligonucleotide can be of any length depending on the number ofdifferent amino acids that must be encoded. In the present example a DNAoligo of 12 nucleotides is used.

The content of all the wells are hereafter pooled, and then split intowells on a new microtitre plate. Each well on the new plate will nowcontain all the different species of bi-functional molecules generatedby the above reactions. A new round of synthesis (round 2) that issimilar to round 1 can hereafter be applied: first an amino acid isadded to each well, one species of amino acid for each well, and linkedto the amino group of the amino acids of the bi-functional molecules inthe well (instead of linking to the amino group of the linker as inround 1). Likewise, different DNA oligos are added to each well andlinked to the DNA-portion of the bi-functional molecules in the wells.Like in the first round, the oligos in each well have differentsequences so that each newly added amino acid is uniquely encoded by thesequence of the newly added oligo. Each well now contains abi-functional molecule consisting of a di-peptide (two amino acids)linked to an oligonucleotide through a linker molecule. Theoligonucleotide, which in the present example is now 24 nucleotideslong, uniquely identifies the di-peptide to which it is linked. Thesequence of the first 12 nucleotides encodes the species of the firstamino acid in the di-peptide and the sequence of the next 12 nucleotidesencodes the identity of the second amino acid.

The amino acids used in the second round can be from the same repertoireas used in round 1, or it can be a different set of amino acids or othercompletely different types of molecule fragments. In the present examplethey are from a different repertoire of amino acids in order to increasethe diversity of the molecules.

The desired coupling reactions of the reactive groups are ensured byprotecting and de-protecting the relevant reactive groups of themolecules during the process.

The content of each well is hereafter pooled as after round 1. A newround of synthesis can hereafter be applied by splitting the pooledbi-functional molecules into wells on a new plate and repeating round 2,which would create bi-functional molecules consisting of tri-peptides(or other kinds of compounds made up of three molecule fragments)attached to 36-mer identifier oligos through a linker. Principally themolecules could be increased in size and diversity by applying newrounds of synthesis until the desired compound is obtained. In thepresent example the synthesis is terminated after round 2. The pool ofbi-functional molecules that is obtained will have a diversity thatdepends on the number of amino acids (or other molecule fragments) usedfor the synthesis in round 1 and 2. If 1000 different molecule fragmentsare used in both rounds the number of different bi-functional moleculesin the pool will be one million (1000 times 1000). The length of theattached oligos (which is in this example 24 nucleotides) is more thanenough to uniquely encode the one million different compounds formed.

Thus, using this split and mix approach a repertoire of bi-functionalcarrier molecules have been generated. This repertoire of bi-functionalcarrier molecules are then used in Stage 2 to generate, through aDNA-templating approach, an even bigger repertoire of bi-functionalmolecules.

Stage 2:

The next stage of the library synthesis uses the bi-functional carriermolecules generated in stage 1 in DNA templated synthesis reactions,which essentially links together the bi-functional carrier moleculesprovided by stage 1 in different combinations. The molecules generatedthis way are all uniquely identifiably by attached templates that encodethe bi-functional carrier molecules that were combined by the reaction.One advantage of the templated carrier synthesis reaction is that thetemplate, in addition to being a unique identifier that encodes thecarrier molecules that are linked together by the reaction, also bringsthe molecules into close proximity and thereby essentially eliminatesthe likelihood of reactions to occur with other template-carriercomplexes. Another advantage, important for the present invention, isthat the DNA template can easily be amplified (e.g. by PCR usingappropriate primer binding sites included in the template) and serve forthe amplification of molecules that are isolated in a screening assay,even if only a small number of each molecule is present in the isolatedsamples. The amplification allows for the execution of further rounds ofscreening and selection, until molecules with desired characteristics(e.g., affinity, specificity, or catalytic activity) are identified.

Several different methods of templated synthesis have been proposed asdescribed in the background of the invention. In principle any of thesemethods could be used to carry out the reaction set in stage 2. In oneembodiment of the invention the templated synthesis is done using themethod described below and illustrated in FIG. 2.

Example of Template Directed Synthesis Using Bi-Functional CarrierMolecules Generated in Stage 1 above:

The following example is a modification of the method described byWalder et al (Walder, J. A. et al. (1979) Proc. Natl. Acad. Sci., 76;51-55). FIG. 2 shows an example of templated synthesis using templatesthat are complementary to pairs of bi-functional carrier moleculesgenerated in step 1. Thus, two bi-functional carrier molecules, eachcomprising an encoded molecule generated in Stage 1 above (thus, in thisexample, a di-peptide) that is linked to a unique DNA oligonucleotideidentifier (in this example, a 24-nucleotide DNA oligo), are broughtinto close proximity through hybridisation of the carriers' DNA portionto a complementary DNA template. The sequence of the DNA template thusdetermines which carrier molecules bind to the template. A reactionbetween the reactive groups of the two carriers hybridised to the sametemplate may now be induced. In the present example, the design of themolecule fragments in Stage 1 led to carrier molecules that can reactthrough an acylation reaction in Stage 2. Thus, an acyl transferreaction (see e.g., FIGS. 3 and 6A) leads to the transfer of the encodedmolecule (here, a di-peptide) of one carrier onto the encoded molecule(here, a di-peptide) of the other carrier, resulting in the generationof a bi-functional molecule where the encoded molecule (here, atetra-peptide) is linked, covalently or non-covalently (in this examplenon-covalently) to a template that encodes the combination of thedi-peptides and thus, ultimately encodes the tetrapeptide.

The acyl transfer leaves the donor carrier as an “empty” oligonucleotidewithout encoded molecule. Both the empty donor carrier and the acceptorcarrier molecule (now carrying the full encoded molecule, here thetetrapeptide) may be attached to the DNA template throughout the acyltransfer reaction. In order to keep the encoded molecule (tetrapeptide)physically associated with a DNA that encodes it, the two carriers mustbe linked together, or alternatively the carrier that carries theencoded molecule (the tetra-peptide) must be linked covalently ornon-covalently to the DNA template. Several ways to ensure theassociation of the encoded molecule with a DNA molecule that encodes itcan be envisioned, some of which are shown in the section “alternativemethods for templated synthesis using carriers”.

In a simple embodiment, the link to the DNA template is kept byconducting the subsequent screening of the library under conditions thatdoes not disrupt the hybridisation of the oligonucleotide identifiers ofthe carriers to the template.

In the present invention, as shown in the example in FIG. 2, the carriermolecules used in the templated synthesis in stage 2 originate from therepertoire of different bi-functional di-peptide carrier moleculesgenerated by the reactions in stage 1. These are di-peptides attachedthrough a linker to a 24-mer oligonucleotide, where the first 12nucleotides encode the first amino acid of the di-peptide and the next12 nucleotides encode the second amino acid. A library of DNA templatesis therefore synthesised where each template consists of a codingsequence of 48 nucleotides; the first 24 nucleotides are complementaryto the 24-mer identifier oligonucleotide of one di-peptide carriermolecule from stage 1 and the last 24 nucleotides are complementary toanother 24-mer oligo from the repertoire of carrier molecules. Thus, thesequence of the template encodes which di-peptide carriers can bind andthereby it encodes and uniquely identifies the four amino acidpolypeptides that result from the transfer of one di-peptide onto theother in the acyl transfer reaction. The two carrier molecules fromstage 1 that are encoded by the template can be from the same repertoireor from two different sets of bi-functional carriers. In the presentexample they originate from different repertoires.

If one million different bi-functional carrier molecules were generatedin stage 1 in the example and combined in stage 2 with another set ofone million different bi-functional carriers, the diversity of theresulting library of molecules generated in stage 2 would be 10¹² (1million times 1 million) different four amino acid polypeptides. Ifdifferent repertoires of amino acids and carriers are used in stage 1and 2, respectively, a total of up to 4000 different amino acids wouldbe required to carry out the synthesis as in the present example. If thesame repertoire of amino acids was used only 1000 different amino acidswould be required, but the resulting library would be less complexalthough it would still contain 10¹² different molecules.

During Stage 1, the oligonucleotide identifiers may be linked to thelinker or to the oligonucleotide portion of the nascent bi-functionalmolecule by enzymatic means, e.g. by ligases (e.g. T4 DNA ligase, E.coli DNA ligase, or T7 DNA ligase for double stranded DNA fragments, orT4 RNA ligase for single-stranded DNA fragments), or by chemicalligation. Several methods for chemical ligation are known in the art,such as the 5′-phosphoimidazolid method (Visscher, J., Shwartz, A. W.Journal of Molecular Evolution (1988), 28, 3-6; Zhao, Y., Thorson, J. S.Journal of Organic Chemistry (1998), 63, 7568-7572), or the3′-phosphothioate method (Alvarez et al., Journal of Organic Chemistry(1999), 64, 6319-28; Pirrung et al., Journal of Organic Chemistry (1998)63, 241-46). Other means of ligating together two oligonucleotidesinclude the use of CNBr as a condensating agent for chemical ligation(Sokolova, N. I., et al., FEBS Letters (1988), 232, 153-155; Dolinnaya,N. G. et al., Nucl. Acids. Res. (1993), 21, 5403-5407); reductiveamination between juxtaposed amine and aldehyde groups (Goodwin, J. T.,and Lynn, D. G., J. Am. Chem. Soc. (1992), 114, 9197-9198); disulfidebond formation (e.g. reaction of a thiol and an activated disulfide suchas pyridyl disulfide); reaction between pyrophosphate-activated5′-phosphate and 3′-hydroxyl, to form a phosphodiester bond (Rohatgi,R., et al., J. Am. Chem. Soc. (1996), 118, 3332-3339); a tosyldisplacement reaction (Herrlein, M. K., et al, J. Am. Chem. Soc. (1995),117, 10151-10152); and reaction of 5′-iodonucleoside and3-phosphothioate (Yanzheng, X., and Kool, E. T., Tetrahedron Letters(1997), 38, 5595-5598.

Some of the ligation reactions generate natural phosphodiester bonds,while other ligation reactions generate non-natural bonds between theligated oligonucleotides. Some of the more effective methods for formingnatural phosphodiester bonds utilise activation of a phosphoryl groupwith CNBr, cyanoimidazole or water soluble carbodiimide, described inthe following papers: Wang, E., Yanagawa, H. Biochemistry (1986), 25,7423-7430; Shabarova, Z. A., Biochemie (1988), 70, 1323-1334; Dolinnaya,N. G., Merenkova, I. N., Shabarova, Z. A. Nucleosides Nucleotides(1994), 13, 2169-2183; Kool, E. T. J. Am. Chem. Soc. (1991), 113,6265-6266; Ashley, G. W., Kushlan, D. M. Biochemistry (1991), 30,2927-2933; Luebke, K. J., Dervan, P. B., J. Am. Chem. Soc. (1991), 113,7447-7448; Luebke, K. J., Dervan, P. B. Nucl. Acids Res. (1992), 20,3005-3009; Gao, H., et al., Bioconjugate Chem. (1994), 5, 445-453. Inparticular, 3′-OH and 5′-phosphate groups, or functional derivativesthereof, have been used to ligate together oligonucleotides, to form anatural phosphodiester bond. Other examples of reactions that may beused to link together identifiers, and the bonds resulting from thosereactions, are shown in FIGS. 6 and 7.

The standard phosphoramidite method for oligonucleotide synthesis mayalso be applied for chemical ligation. For both enzymatic and chemicalligation it is preferable that the DNA fragments to be ligated are ondouble-stranded form and with overhangs. In stage 1 synthesis, theidentifier may be attached as a double stranded oligonucleotide or asingle stranded oligonucleotide, and likewise, the identifier of thenascent bi-functional molecule may be on double- or single-strandedform. The incoming oligonucleotide may be attached by ligation (chemicalor enzymatic) of one or two strands, or may be attached by non-covalentinteraction of the incoming identifier and the oligonucleotide of thebi-functional molecule, e.g. by annealing of complementaryoligonucleotide regions of the incoming oligonucleotide and thebi-functional molecule, or by a third oligonucleotide and the incomingidentifier/bi-functional molecule.

In a preferred embodiment the oligonucleotide identifiers are notde-protected (after the phosphoramidite synthesis of individualidentifiers), prior to their linking in the Stage 1 synthesis. This willallow conditions to be applied in the linking of molecule fragments andlinking of oligonucleotide identifiers during the stage 1 process thatcould not otherwise easily have been applied, such as the use of certainorganic solvents. As an example, if the reaction that is used forlinking two unit identifiers together during a stage 1 synthesis requirerelatively strong acidic conditions, and the use of an organic solventsuch as DMF, it may be desirable to use oligonucleotides, for which thenucleic acid bases have not been deprotected, as this will make themless vulnerable to acidic conditions, and also, will make them moresoluble in organic solvents such as DMF. Then, after the stage 1synthesis of the carrier molecule, the oligonucleotide component may bedeprotected, to allow efficient sequence specific interaction betweenthe carrier and the template in a stage 2 synthesis.

Finally, the ligation of unit identifiers may lead to linear as well asbranched products. An alternative method for performing stage 1synthesis involves the hybridisation between the bi-functional moleculeand the incoming oligonucleotide, where then an extension reaction isperformed using e.g. a polymerase to extend from the 3′-end of thebi-functional molecule, to add an identifier sequence (Freskård et al.,WO 2004/039825 A2, Enzymatic Encoding). Polymerases that may be used inthis approach include Reverse transcriptase, DNA polymerase, RNApolymerase, Taq polymerase, Pfu polymerase, Vent polymerase, Klenowfragment, and many others.

During templated synthesis in stage 2 the oligonucleotide portions ofthe carrier molecules may be ligated together by enzymatic or chemicalligation.

During both stage 1 and stage 2, the bi-functional molecules may beimmobilised to solid support, allowing simple and efficient removal ofreagents and by-products, as well as allowing the use of organicsolvents that might otherwise precipitate molecule fragments,oligonucleotides or bi-functional molecules.

A wide range of conditions may be applied to mediate the reactionbetween the nascent bi-functional molecule and the molecule fragment.These include the addition of catalysts, base, acid, or reagents thattake part in the reaction. The latter may react with both the nascentbi-functional molecule and the molecule fragment, and thus may end up asa bridge or linker that links the nascent bi-functional molecule and themolecule fragment. Such linking moeties include di-carboxylic acids(where for example the reactive groups to become linked are two amines),or di-amines (where two carboxylic acids become linked through thelinking moiety).

Reactive groups of the present invention include aldehydes, hydroxyls,isocyanate, thiols, amines, esters, thioesters, carboxylic acids, triplebonds, double bonds, ethers, acid chlorides, phosphates, imidazoles,halogenated aromatic rings, any precursors thereof, or any protectedreactive groups. Examples of reactive groups that can be employed duringstage 1, and the bonds resulting from these reactions, are shown inFIGS. 6 and 7. Reactions that may be employed during stage 1 synthesisinclude acylation (formation of amide, pyrazolone, isoxazolone,pyrimidine, comarine, quinolinon, phthalhydrazide, diketopiperazine,benzodiazepinone, and hydantoin), alkylation, vinylation, disulfideformation, Wittig reaction, Horner-Wittig-Emmans reaction, arylation(formation of biaryl or vinylarene), condensation reactions,cycloadditions ([2+4], [3+2]), addition to carbon-carbon multiplebonds,cycloaddition to multiple bonds, addition to carbon-hetero multiplebonds, nucleophilic aromatic substitution, transition metal catalyzedreactions, as well as the reaction types listed below, and may involveformation of ethers, thioethers, secondary amines, tertiary amines,beta-hydroxy ethers, beta-hydroxy thioethers, beta-hydroxy amines,beta-amino ethers, amides, thioamides, oximes, sulfonamides, di- andtri-functional compounds, substituted aromatic compounds, vinylsubstituted aromatic compounds, alkyn substituted aromatic compounds,biaryl compounds, hydrazines, hydroxylamine ethers, substitutedcycloalkenes, substituted cyclodienes, substituted 1, 2, 3 triazoles,substituted cycloalkenes, beta-hydroxy ketones, beta-hydroxy aldehydes,vinyl ketones, vinyl aldehydes, substituted alkenes, substitutedalkenes, substituted amines, and many others.

Molecule Fragments, Identifiers and Reagents.

In stage 1 synthesis, any number of molecule fragments may be linked tothe nascent bi-functional molecule during a synthesis round. Thus, 0, 1,2, 3 or more molecule fragments may be linked to the nascentbi-functional molecule in a given synthesis round. In the same synthesisround an identifier encoding the molecule fragment(s) that were linkedto the nascent bi-functional molecule is linked to the nascentoligonucleotide of the bi-functional molecule. Thus, one identifier mayencode a combination of several molecule fragments, and also, may encodemolecule fragment(s), reagent(s), catalyst(s) and conditions employed.In such cases, two different identifiers may encode the same moleculefragment but different reaction conditions. If desired, more than oneidentifier may be linked to the nascent oligonucleotide. For example, iftwo molecule fragments are reacted with the nascent bi-functionalmolecule, one may choose to also link two identifiers to the nascentoligonucleotide. However, in most cases one identifier is used to encodeall the molecule fragments added in a given synthesis round.

In a given synthesis round, the attachment of the molecule fragment(s)to the nascent bi-functional molecule can be performed before,simultaneously with, or after the attachment of the identifier(s)encoding said molecule fragments.

In stage 2 synthesis, any number of carriers may be addedsimultaneously, in order to transfer 0, 1, 2, 3, 4 or more moleculefragments to the nascent bi-functional molecule. Alternatively, carriersare added sequentially or partly sequentially (e.g. two carriers at atime), and therefore, the molecule fragments of the final bi-functionalmolecule are made up of molecule fragments that have been transferredsimultaneously, partly sequentially or fully sequentially (i.e., one ata time).

During the templated synthesis of stage 2, the molecule fragments areattached to the oligonucleotide identifiers prior to reaction betweencarriers. During the templated synthesis, the molecule fragment of onecarrier is transferred to another carrier. This may involve a directtransfer in which the reaction between two reactive groups directlyleads to a transfer of one molecule fragment from one carrier to another(FIG. 3A). Reactions that mediate the direct transfer of one moleculefragment from one carrier to another carrier include the reactionslisted in FIG. 6. Alternatively, a transfer may involve first a reactionbetween the reactive groups, followed by cleavage of the bond linkingone molecule fragment to the carrier, which will result in the transferof that molecule fragment onto the other carrier (FIG. 3B). Types ofdirect and indirect transfer reactions, as well as the bonds resultingfrom the reaction of reactive groups, are shown in FIGS. 6 and 7. Anumber of reactions for stage 1 and stage 2 synthesis are listed below.

In certain embodiments the molecule fragments remain associated withboth carriers after the reaction step of Stage 2, i.e., it is not arequirement in the present invention that the molecule fragments aretransferred from one carrier to another.

Reaction Conditions Compatible with Stage 1 and Stage 2 Synthesis ofBifunctional Molecules.

Stage 1 and stage 2 reactions can occur in aqueous or non-aqueous (i.e.,organic) solutions, or a mixture of one or more aqueous and non-aqueoussolutions. In aqueous solutions, reactions can be performed at pH rangesfrom about 2 to about 12, or preferably from about 2 to about 10, ormore preferably from about 4 to about 10. The reactions used inDNA-templated chemistry preferably should not require very basicconditions (e.g., pH>13, pH>10), or very acidic conditions (e.g., pH <1,pH <2, pH <4), because extreme conditions may lead to degradation ormodification of the nucleic acid template and/or encoded molecule beingsynthesized. The aqueous solution can contain one or more inorganicsalts, including, but not limited to, NaCl, Na₂SO₄, KCl, Mg⁺², Mn⁺²,etc., at various concentrations.

Organic solvents suitable for stage 1 and stage 2 reactions include, butare not limited to, methylene chloride, chloroform, dimethylformamide,and organic alcohols, including methanol and ethanol. To permitquantitative dissolution of reaction components in organic solvents,quaternized ammonium salts, such as, for example, long chaintetraalkylammonium salts, can be added (Jost et al. (1989) NUCLEIC ACIDSRES. 17:2143; Melnikov et al. (1999) LANGMUIR 15: 1923-1928).

Stage 1 or stage 2 reactions may require a catalyst, such as, forexample, homogeneous, heterogeneous, phase transfer, and asymmetriccatalysis. In other embodiments, a catalyst is not required. Thepresence of additional, accessory reagents not linked to a nucleic acidare preferred in some embodiments. Useful accessory reagents caninclude, for example, oxidizing agents (e.g., NaIO₄; reducing agents(e.g., NaCNBH₃); activating reagents (e.g., EDC, NHS, and sulfo-NHS);transition metals such as nickel (e.g., Ni(NO₃)₂), rhodium (e.g. RhCl₃),ruthenium (e.g. RuCl₃), copper (e.g. Cu(NO₃)₂), cobalt (e.g. COCl₂),iron (e.g. Fe,(NO₃)₃), osmium (e.g. OsO₄), titanium (e.g. TiCl₄ ortitanium tetraisopropoxide), palladium (e.g. NaPdCl₄), or Ln; transitionmetal ligands (e.g., phosphines, amines, and halides); Lewis acids; andLewis bases.

Reaction conditions preferably are optimized to suit the nature of thereactive units and oligonucleotides used.

Reaction Types Compatible with Stage 1 and Stage 2 Synthesis.

Known chemical reactions for synthesizing polymers, small molecules, orother chemical compounds can be used in stage 1 and stage 2 synthesisreactions. Thus, reactions such as those listed in March's AdvancedOrganic Chemistry, Organic Reactions, Organic Syntheses, organic textbooks, journals such as Journal of the American Chemical Society,Journal of Organic Chemistry, Tetrahedron, etc., and Carruther's SomeModern Methods of Organic Chemistry can be used. The chosen reactionspreferably are compatible with nucleic acids such as DNA or RNA or arecompatible with the modified nucleic acids used as the template.

Reactions useful in stage 1 and stage 2 synthesis include, for example,substitution reactions, carbon-carbon bond forming reactions,elimination reactions, acylation reactions, and addition reactions. Anillustrative but not exhaustive list of aliphatic nucleophilicsubstitution reactions useful in the present invention includes, forexample, SN2 reactions, SNI reactions, S_(N)i reactions, allylicrearrangements, nucleophilic substitution at an aliphatic trigonalcarbon, and nucleophilic substitution at a vinylic carbon.

Specific aliphatic nucleophilic substitution reactions with oxygennucleophiles include, for example, hydrolysis of alkyl halides,hydrolysis of gen-dihalides, hydrolysis of 1,1,1-trihalides, hydrolysisof alkyl esters or inorganic acids, hydrolysis of diazo ketones,hydrolysis of acetal and enol ethers, hydrolysis of epoxides, hydrolysisof acyl halides, hydrolysis of anhydrides, hydrolysis of carboxylicesters, hydrolysis of amides, alkylation with alkyl halides (WilliamsonReaction), epoxide formation, alkylation with inorganic esters,alkylation with diazo compounds, dehydration of alcohols,transetherification, alcoholysis of epoxides, alkylation with oniumsalts, hydroxylation of silanes, alcoholysis of acyl halides,alcoholysis of anhydrides, esterfication of carboxylic acids,alcoholysis of carboxylic esters (transesterfication), alcoholysis ofamides, alkylation of carboxylic acid salts, cleavage of ether withacetic anhydride, alkylation of carboxylic acids with diazo compounds,acylation of carboxylic acids with acyl halides; acylation of carboxylicacids with carboxylic acids, formation of oxonium salts, preparation ofperoxides and hydroperoxides, preparation of inorganic esters (e.g.,nitrites, nitrates, sulfonates), preparation of alcohols from amines,and preparation of mixed organic-inorganic anhydrides.

Specific aliphatic nucleophilic substitution reactions with sulfurnucleophiles, which tend to be better nucleophiles than their oxygenanalogs, include, for example, attack by SH at an alkyl carbon to formthiols, attack by S at an alkyl carbon to form thioethers, attack by SHor SR at an acyl carbon, formation of disulfides, formation of Buntesalts, alkylation of sulfuric acid salts, and formation of alkylthiocyanates.

Aliphatic nucleophilic substitution reactions with nitrogen nucleophilesinclude, for example, alkylation of amines, N-arylation of amines,replacement of a hydroxy by an amino group, transamination,transamidation, alkylation of amines with diazo compounds, animation ofepoxides, amination of oxetanes, amination of aziridines, amination ofalkanes, formation of isocyanides, acylation of amines by acyl halides,acylation of amines by anhydrides, acylation of amines by carboxylicacids, acylation of amines by carboxylic esters, acylation of amines byamides, acylation of amines by other acid derivatives, N-alkylation orN-arylation of amides and imides, N-acylation of amides and imides,formation of aziridines from epoxides, formation of nitro compounds,formation of azides, formation of isocyanates and isothiocyanates, andformation of azoxy compounds.

Aliphatic nucleophilic substitution reactions with halogen nucleophilesinclude, for example, attack at an alkyl carbon, halide exchange,formation of alkyl halides from esters of sulfuric and sulfonic acids,formation of alkyl halides from alcohols, formation of alkyl halidesfrom ethers, formation of halohydrins from epoxides, cleavage ofcarboxylic esters with lithium iodide, conversion of diazo ketones toalpha-halo ketones, conversion of amines to halides, conversion oftertiary amines to cyanamides (the von Braun reaction), formation ofacyl halides from carboxylic acids, and formation of acyl halides fromacid derivatives.

Aliphatic nucleophilic substitution reactions using hydrogen as anucleophile include, for example, reduction of alkyl halides, reductionof tosylates, other sulfonates, and similar compounds, hydrogenolysis ofalcohols, hydrogenolysis of esters (Barton-McCombie reaction),hydrogenolysis of nitriles, replacement of alkoxyl by hydrogen,reduction of epoxides, reductive cleavage of carboxylic esters,reduction of a C—N bond, desulfurization, reduction of acyl halides,reduction of carboxylic acids, esters, and anhydrides to aldehydes, andreduction of amides to aldehydes.

Although certain carbon nucleophiles may be too nucleophilic and/orbasic to be used in certain embodiments of the invention, aliphaticnucleophilic substitution reactions using carbon nucleophiles include,for example, coupling with silanes, coupling of alkyl halides (the Wurtzreaction), the reaction of alkyl halides and sulfonate esters with GroupI (I A), and II (II A) organometallic reagents, reaction of alkylhalides and sulfonate esters with organocuprates, reaction of alkylhalides and sulfonate esters with other organometallic reagents; allylicand propargylic coupling with a halide substrate, coupling oforganometallic reagents with esters of sulfuric and sulfonic acids,sulfoxides, and sulfones, coupling involving alcohols, coupling oforganometallic reagents with carboxylic esters, coupling oforganometallic reagents with compounds containing an esther linkage,reaction of organometallic reagents with epoxides, reaction oforganometallics with aziridine, alkylation at a carbon bearing an activehydrogen, alkylation of ketones, nitriles, and carboxylic esters,alkylation of carboxylic acid salts, alkylation at a position alpha to aheteroatom (alkylation of 1,3-dithianes), alkylation ofdihydro-1,3-oxazine (the Meyers synthesis of aldehydes, ketones, andcarboxylic acids), alkylation with trialkylboranes, alkylation at analkynyl carbon, preparation of nitriles, direct conversion of alkylhalides to aldehydes and ketones, conversion of alkyl halides, alcohols,or alkanes to carboxylic acids and their derivatives, the conversion ofacyl halides to ketones with organometallic compounds, the conversion ofanhydrides, carboxylic esters, or amides to ketones with organometalliccompounds, the coupling of acyl halides, acylation at a carbon bearingan active hydrogen, acylation of carboxylic esters by carboxylic esters(the Claisen and Dieckmann condensation), acylation of ketones andnitriles with carboxylic esters, acylation of carboxylic acid salts,preparation of acyl cyanides, and preparation of diazo ketones, ketonicdecarboxylation.

Reactions which involve nucleophilic attack at a sulfonyl sulfur atommay also be used in the present invention and include, for example,hydrolysis of sulfonic acid derivatives (attack by OH), formation ofsulfonic esters (attack by OR), formation of sulfonamides (attack bynitrogen), formation of sulfonyl halides (attack by halides), reductionof sulfonyl chlorides (attack by hydrogen), and preparation of sulfones(attack by carbon).

Aromatic electrophilic substitution reactions may also be used in stage1 and stage 2 synthesis schemes. Hydrogen exchange reactions areexamples of aromatic electrophilic substitution reactions that usehydrogen as the electrophile. Aromatic electrophilic substitution,reactions which use nitrogen electrophiles include, for example,nitration and nitro-dehydrogenation, nitrosation ofnitroso-de-hydrogenation, diazonium coupling, direct introduction of thediazonium group, and amination or amino-dehydrogenation. Reactions ofthis type with sulfur electrophiles include, for example, sulfonation,sulfo-dehydrogenation, halosulfonation, halosulfo-dehydrogenation,sulfurization, and sulfonylation. Reactions using halogen electrophilesinclude, for example, halogenation, and halo-dehydrogenation. Aromaticelectrophilic substitution reactions with carbon electrophiles include,for example, Friedel-Crafts alkylation, alkylation,alkyl-dehydrogenation, Friedel-Crafts arylation (the Scholl reaction),Friedel-Crafts acylation, formylation with disubstituted formamides,formylation with zinc cyanide and HCl (the Gatterman reaction),formylation with chloroform (the Reimer-Tiemami reaction), otherformylations, formyl-dehydrogenation, carboxylation with carbonylhalides, carboxylation with carbon dioxide (the Kolbe-Schmitt reaction),amidation with isocyanates, N-alkylcarbamoyl-dehydrogenation,hydroxyalkylation, hydroxyalkyl-dehydrogenation, cyclodehydration ofaldehydes and ketones, haloalkylation, halo-dehydrogenation,aminoalkylation, aminoalkylation, dialkylaminoalkylation,dialkylamino-dehydrogenation, thioalkylation, acylation with nitriles(the Hoesch reaction), cyanation, and cyano-de hydrogenation. Reactionsusing oxygen electrophiles include, for example, hydroxylation andhydroxy-dehydrogenation.

Rearrangement reactions include, for example, the Fries rearrangement,migration of a nitro group, migration of a nitroso group (theFischer-Hepp Rearrangement), migration of an arylazo group, migration ofa halogen (the Orton rearrangement), migration of an alkyl group, etc.Other reaction on an aromatic ring include the reversal of aFriedel-Crafts alkylation, decarboxylation of aromatic aldehydes,decarboxylation of aromatic acids, the Jacobsen reaction, deoxygenation,desulfonation, hydro-desulfonation, dehalogenation,hydro-dehalogenation, and hydrolysis of organometallic compounds.

Aliphatic electrophilic substitution reactions are also useful.Reactions using the S_(E)I, S_(E)2 (front), S_(E)2 (back), S_(E)i,addition-elimination, and cyclic mechanisms can be used in the presentinvention. Reactions of this type with hydrogen as the leaving groupinclude, for example, hydrogen exchange (deuterio-de-hydrogenation,deuteriation), migration of a double bond, and keto-enoltautomerization. Reactions with halogen electrophiles include, forexample, halogenation of aldehydes and ketones, halogenation ofcarboxylic acids and acyl halides, and halogenation of sulfoxides andsulfones. Reactions with nitrogen electrophiles include, for example,aliphatic diazonium coupling, nitrosation at a carbon bearing an activehydrogen, direct formation of diazo compounds, conversion of amides toalpha-azido amides, direct amination at an activated position, andinsertion by nitrenes. Reactions with sulfur or selenium electrophilesinclude, for example, sulfenylation, sulfonation, and selenylation ofketones and carboxylic esters. Reactions with carbon electrophilesinclude, for example, acylation at an aliphatic carbon, conversion ofaldehydes to beta-keto esters or ketones, cyanation,cyano-de-hydrogenation, alkylation of alkanes, the Stork enaminereaction, and insertion by carbenes. Reactions with metal electrophilesinclude, for example, metalation with organometallic compounds,metalation with metals and strong bases, and conversion of enolates tosilyl enol ethers. Aliphatic electrophilic substitution reactions withmetals as leaving groups include, for example, replacement of metals byhydrogen, reactions between organometallic reagents and oxygen,reactions between organometallic reagents and peroxides, oxidation oftrialkylboranes to borates, conversion of Grignard reagents to sulfurcompounds, halo-demetalation, the conversion of organometallic compoundsto amines, the conversion of organometallic compounds to ketones,aldehydes, carboxylic esters and amides, cyano-de-metalation,transmetalation with a metal, transmetalation with a metal halide,transmetalation with an organometallic compound, reduction of alkylhalides, metallo-de-halogenation, replacement of a halogen by a metalfrom an organometallic compound, decarboxylation of aliphatic acids,cleavage of alkoxides, replacement of a carboxyl group by an acyl group;basic cleavage of beta-keto esters and beta-diketones, haloformreaction, cleavage of non-enolizable ketones, the Haller-Bauer reaction,cleavage of alkanes, decyanation, and hydro-de-cyanation. Electrophilicsubstitution reactions at nitrogen include, for example, diazotization,conversion of hydrazines to azides, N-nitrosation,N-nitroso-de-hydrogenation, conversion of amines to azo compounds,N-halogenation, N-halo-de-hydrogenation, reactions of amines with carbonmonoxide, and reactions of amines with carbon dioxide.

Aromatic nucleophilic substitution reactions may also be used in thepresent invention. Reactions proceeding via the S_(N)Ar mechanism, theS_(N)1 mechanism, the benzyne mechanism, the S_(RN)1 mechanism, or othermechanism, for example, can be used. Aromatic nucleophilic substitutionreactions with oxygen nucleophiles include, for example,hydroxy-de-halogenation, alkali fusion of sulfonate salts, andreplacement of OR or OAr. Reactions with sulfur nucleophiles include,for example, replacement by SH or SR. Reactions using nitrogennucleophiles include, for example, replacement by NH₂, NHR, or NR₂, andreplacement of a hydroxy group by an amino group: Reactions with halogennucleophiles include, for example, the introduction halogens. Aromaticnucleophilic substitution reactions with hydrogen as the nucleophileinclude, for example, reduction of phenols and phenolic esters andethers, and reduction of halides and nitro compounds. Reactions withcarbon nucleophiles include, for example, the Rosenmund-von Braunreaction, coupling of organometallic compounds with aryl halides,ethers, and carboxylic esters, arylation at a carbon containing anactive hydrogen, conversions of aryl substrates to carboxylic acids,their derivatives, aldehydes, and ketones, and the Ullmann reaction.Reactions with hydrogen as the leaving group include, for example,alkylation, arylation, and amination of nitrogen heterocycles. Reactionswith N₂ ⁺ as the leaving group include, for example,hydroxy-de-diazoniation, replacement by sulfur-containing groups,iodo-de-diazoniation, and the Schiemann reaction. Rearrangementreactions include, for example, the von Richter rearrangement, theSommelet-Hauser rearrangement, rearrangement of aryl hydroxylamines, andthe Smiles rearrangement. Reactions involving free radicals can also beused, although the free radical reactions used in nucleotide-templatedchemistry should be carefully chosen to avoid modification or cleavageof the nucleotide template. With that limitation, free radicalsubstitution reactions can be used in the present invention. Particularfree radical substitution reactions include, for example, substitutionby halogen, halogenation at an alkyl carbon, allylic halogenation,benzylic halogenation, halogenation of aldehydes, hydroxylation at analiphatic carbon, hydroxylation at an aromatic carbon, oxidation ofaldehydes to carboxylic acids, formation of cyclic ethers, formation ofhydroperoxides, formation of peroxides, acyloxylation,acyloxy-de-hydrogenation, chlorosulfonation, nitration of alkanes,direct conversion of aldehydes to amides, amidation and amination at analkyl carbon, simple coupling at a susceptible position, coupling ofalkynes, arylation of aromatic compounds by diazonium salts, arylationof activated alkenes by diazonium salts (the Meerwein arylation),arylation and alkylation of alkenes by organopalladium compounds (theHeck reaction), arylation and alkylation of alkenes by vinyltincompounds (the Stille reaction), alkylation and arylation of aromaticcompounds by peroxides, photochemical arylation of aromatic compounds,alkylation, acylation, and carbalkoxylation of nitrogen heterocy'cles.Particular reactions in which N₂ ⁺ is the leaving group include, forexample, replacement of the diazonium group by hydrogen, replacement ofthe diazonium group by chlorine or bromine, nitro-de-diazoniation,replacement of the diazonium group by sulfur-containing groups, aryldimerization with diazonium salts, methylation of diazonium salts,vinylation of diazonium salts, arylation of diazonium salts, andconversion of diazonium salts to aldehydes, ketones, or carboxylicacids. Free radical substitution reactions with metals as leaving groupsinclude, for example, coupling of Grignard reagents, coupling ofboranes, and coupling of other organometallic reagents. Reaction withhalogen as the leaving group are included. Other free radicalsubstitution reactions with various leaving groups include, for example,desulfurization with Raney Nickel, conversion of sulfides toorganolithium compounds, decarboxylative dimerization (the Kolbereaction), the Hunsdiecker reaction, decarboxylative allylation, anddecarbonylation of aldehydes and acyl halides.

Reactions involving additions to carbon-carbon multiple bonds are alsoused in the stage 1 and stage 2 synthesis schemes. Any mechanism may beused in the addition reaction including, for example, electrophilicaddition, nucleophilic addition, free radical addition, and cyclicmechanisms. Reactions involving additions to conjugated systems can alsobe used. Addition to cyclopropane rings can also be utilized. Particularreactions include, for example, isomerization, addition of hydrogenhalides, hydration of double bonds, hydration of triple bonds, additionof alcohols, addition of carboxylic acids, addition of H₂S and thiols,addition of ammonia and amines, addition of amides, addition ofhydrazoic acid, hydrogenation of double and triple bonds, otherreduction of double and triple bonds, reduction of the double and triplebonds of conjugated systems, hydrogenation of aromatic rings, reductivecleavage of cyclopropanes, hydroboration, other hydrometalations,addition of alkanes, addition of alkenes and/or alkynes to alkenesand/or alkynes (e.g., pi-cation cyclization reactions,hydro-alkenyl-addition), ene reactions, the Michael reaction, additionof organometallics to double and triple bonds not conjugated tocarbonyls, the addition of two alkyl groups to an alkyne, 1,4-additionof organometallic compounds to activated double bonds, addition ofboranes to activated double bonds, addition of tin and mercury hydridesto activated double bonds, acylation of activated double bonds and oftriple bonds, addition of alcohols, amines, carboxylic esters,aldehydes, etc., carbonylation of double and triple bonds,hydrocarboxylation, hydroformylation, addition of aldehydes, addition ofHCN, addition of silanes, radical addition, radical cyclization,halogenation of double and triple bonds (addition of halogen, halogen),halolactonization, halolactamization, addition of hypohalous acids andhypohalites (addition of halogen, oxygen), addition of sulfur compounds(addition of halogen, sulfur), addition of halogen and an amino group(addition of halogen, nitrogen), addition of NOX and NO₂X (addition ofhalogen, nitrogen), addition of XN₃ (addition of halogen, nitrogen),addition of alkyl halides (addition of halogen, carbon), addition ofacyl halides (addition of halogen, carbon), hydroxylation (addition ofoxygen, oxygen) (e.g., asymmetric dihydroxylation reaction with OSO₄),dihydroxylation of aromatic rings, epoxidation (addition of oxygen,oxygen) (e.g., Sharpless asymmetric epoxidation), photooxidation ofdienes (addition of oxygen, oxygen), hydroxysulfenylation (addition ofoxygen, sulfur), oxyamination (addition of oxygen, nitrogen),diamination (addition of nitrogen, nitrogen), formation of aziridines(addition of nitrogen), aminosulferiylation (addition of nitrogen,sulfur), acylacyloxylation and acylamidation (addition of oxygen, carbonor nitrogen, carbon), 1,3-dipolar addition; (addition of oxygen,nitrogen, carbon), Diels-Alder reaction, heteroatom Diels-Alderreaction, all carbon 3+2 cycloadditions, dimerization of alkenes, theaddition of carbenes and carbenoids to double and triple bonds,trimerization and tetramerization of alkynes, and other cycloadditionreactions.

In addition to reactions involving additions to carbon-carbon multiplebonds, addition reactions to carbon-hetero multiple bonds can be used innucleotide-templated chemistry. Exemplary reactions include, forexample, the addition of water to aldehydes and ketones (formation ofhydrates), hydrolysis of carbon-nitrogen double bond, hydrolysis ofaliphatic nitro compounds, hydrolysis of nitrites, addition of alcoholsand thiols to aldehydes and ketones, reductive alkylation of alcohols,addition of alcohols to isocyanates, alcoholysis of nitrites, formationof xanthates, addition of H₂S and thiols to carbonyl compounds,formation of bisulfite addition products, addition of amines toaldehydes and ketones, addition of amides to aldehydes, reductivealkylation of ammonia or amines, the Mannich reaction, the addition ofamines to isocyanates, addition of ammonia or amines to nitrites,addition of amines to carbon disulfide and carbon dioxide, addition ofhydrazine derivative to carbonyl compounds, formation of oximes,conversion of aldehydes to nitrites, formation of gem-dihalides fromaldehydes and ketones, reduction of aldehydes and ketones to alcohols,reduction of the carbon-nitrogen double bond, reduction of nitrites toamines, reduction of nitrites to aldehydes, addition of Grignardreagents and organolithium reagents to aldehydes and ketones, additionof other organometallics to aldehydes and ketones, addition oftrialkylallylsilanes to aldehydes and ketones, addition of conjugatedalkenes to aldehydes (the Baylis-Billmah reaction), the Reformatskyreaction, the conversion of carboxylic acid salts to ketones withorganometallic compounds, the addition of Grignard reagents to acidderivatives, the addition of Organometallic compounds to CO₂ and CS₂,addition of organometallic compounds to C═N compounds, addition ofcarbenes and diazoalkanbs to C═N compounds, addition of Grignardreagents to nitriles and isocyanates, the Aldol reaction, MukaiyamaAldol and related reactions, Aldol-type reactions between carboxylicesters or amides and aldehydes or ketones, the Knoevenagel reaction(e.g., the Nef reaction, the Favorskii reaction), the Petersonalkenylation reaction, the addition of active hydrogen compounds to CO₂and CS₂, the Perkin reaction, Darzens glycidic ester condensation, theTollens reaction, the Wittig reaction, the Tebbe alkenylation, thePetasis alkenylation, alternative alkenylations, the Thorpe reaction,the Thorpe-Ziegler reaction, addition of silanes, formation ofcyanohydrins, addition of HCN to C═N and C—N bonds, the Prins reaction,the benzoin condensation, addition of radicals to C═O, C═S, C═Ncompounds, the Ritter reaction, acylation of aldehydes and ketones,addition of aldehydes to aldehydes, the addition of isocyanates toisocyanates (formation of carbodiimides), the conversion of carboxylicacid salts to nitriles, the formation of epoxides from aldehydes andketones, the formation of episulfides and episulfones, the formation ofbeta-lactones and oxetanes (e.g., the Paterno-Buchi reaction), theformation of beta-lactams, etc. Reactions involving addition toisocyanides include the addition of water to isocyanides, the Passerinireaction, the Ug reaction, and the formation of metalated aldimines.

Elimination reactions, including alpha, beta, and gamma eliminations, aswell as extrusion reactions, can be performed using nucleotide-templated chemistry, although the strength of the reagents and conditionsemployed should be considered. Preferred elimination reactions includereactions that go by El, E2, ElcB, or E2C mechanisms. Exemplaryreactions include, for example, reactions in which hydrogen is removedfrom one side (e.g., dehydration of alcohols, cleavage of ethers toalkenes, the Chugaev reaction, ester decomposition, cleavage ofquarternary ammonium hydroxides, cleavage of quaternary ammonium saltswith strong bases, cleavage of amine oxides, pyrolysis of keto-ylids,decomposition of toluene-p-sulfonylhydrazones, cleavage of sulfoxides,cleavage of selenoxides, cleavage of sulformes, dehydrogalogenation ofalkyl halides, dehydrohalogenation of acyl halides, dehydrohalogenationof sulfonyl halides, elimination of boranes, conversion of alkenes toalkynes, decarbonylation of acyl halides), reactions in which neitherleaving atom is hydrogen (e.g., deoxygenation of vicinal diols, cleavageof cyclic thionocarbonates, conversion of epoxides to episulfides andalkenes, the Ramberg-Backlund reaction, conversion of aziridines toalkenes, dehalogenation of vicinal dihalides, dehalogenation ofalpha-halo acyl halides, and elimination of a halogen and a heterogroup), fragmentation reactions

(i.e., reactions in which carbon is the positive leaving group or theelectrofuge, such as, for example, fragmentation of gamma-amino andgamma-hydroxy halides, fragmentation of 1,3-diols, decarboxylation ofbeta-hydroxy carboxylic acids, decarboxylation of (3-lactones,fragmentation of alpha-beta-epoxy hydrazones, elimination of CO frombridged bicyclic compounds, and elimination of CO₂ from bridged bicycliccompounds), reactions in which C═N or C═N bonds are formed (e.g.,dehydration of aldoximes or similar compounds, conversion of ketoximesto nitriles, dehydration of unsubstituted amides, and conversion ofN-alkylformamides to isocyanides), reactions in which C═O bonds areformed (e.g., pyrolysis of beta-hydroxy alkenes), and reactions in whichN═N bonds are formed (e.g., eliminations to give diazoalkenes).Extrusion reactions include, for example, extrusion of N₂ frompyrazolines, extrusion of N₂ from pyrazoles, extrusion of N₂ fromtriazolines, extrusion of CO, extrusion of CO₂, extrusion of SO₂, theStory synthesis, and alkene synthesis by twofold extrusion.

Rearrangements, including, for example, nucleophilic rearrangements,electrophilic rearrangements, prototropic rearrangements, andfree-radical rearrangements, can also be performed using stage 1 andstage 2 synthesis schemes. Both 1,2 rearrangements and non-1,2rearrangements can be performed. Exemplary reactions include, forexample, carbon-to-carbon migrations of R, H, and Ar (e.g.,Wagner-Meerwein and related reactions, the Pinacol rearrangement, ringexpansion reactions, ring contraction reactions, acid-catalyzedrearrangements of aldehydes and ketones, the dienone-phenolrearrangement, the Favorskii rearrangement, the Amdt-Eistert synthesis,homologation of aldehydes, and homologation of ketones),carbon-to-carbon migrations of other groups (e.g., migrations ofhalogen, hydroxyl, amino, etc.; migration of boron; and the Neberrearrangement), carbon-to-nitrogen migrations of R and Ar (e.g., theHofmann rearrangement, the Curtius rearrangement, the Lossenrearrangement, the Schmidt reaction, the Beckman rearrangement, theStieglits rearrangement, and related rearrangements), carbon-to-oxygenmigrations of R and Ar (e.g., the Baeyer-Villiger rearrangement andrearrangment of hydroperoxides), nitrogen-to-carbon, oxygen-to-carbon,and sulfur-to-carbon migration (e.g., the Stevens rearrangement, and theWittig rearrangement), boron-to-carbon migrations (e.g., conversion ofboranes to alcohols (primary or otherwise), conversion of boranes toaldehydes, conversion of boranes to carboxylic acids, conversion ofvinylic boranes to alkenes, formation of alkynes from boranes andacetylides, formation of alkenes from boranes and acetylides, andformation of ketones from boranes and acetylides), electrocyclicrearrangements (e.g., of cydobutenes and 1,3-cyclohexadienes, orconversion of stilbenes to phenanthrenes), sigmatropic rearrangements(e.g., (1,j) sigmatropic migrations of hydrogen, (l,j) sigmatropicmigrations of carbon, conversion of vinylcyclopropanes to cyclopentenes,the Cope rearrangement, the Claisen rearrangement, the Fischer indolesynthesis, (2,3) sigmatropic rearrangements, and the benzidinerearrangement), other cyclic rearrangements (e.g., metathesis ofalkenes, the di-n-methane and related rearrangements, and theHofmann-Loffler and related reactions), and non-cyclic rearrangements(e.g., hydride shifts, the Chapman rearrangement, the Wallachrearrangement, and dybtropic rearrangements).

Oxidative and reductive reactions may also be performed using stage 1and stage 2 synthesis schemes. Exemplary reactions may involve, forexample, direct electron transfer, hydride transfer, hydrogen-atomtransfer, formation of ester intermediates, displacement mechanisms, oraddition-elimination mechanisms. Exemplary oxidations include, forexample, eliminations of hydrogen (e.g., aromatization of six-memberedrings, dehydrogenations yielding carbon-carbon double bonds, oxidationor dehydrogenation of alcohols to aldehydes and ketones, oxidation ofphenols and aromatic amines to quinones, oxidative cleavage of ketones,oxidative cleavage of aldehydes, oxidative cleavage of alcohols,ozonolysis, oxidative cleavage of double bonds and aromatic rings,oxidation of aromatic side chains, oxidative decarboxylation, andbisdecarboxylation), reactions involving replacement of hydrogen byoxygen (e.g., oxidation of methylene to carbonyl, oxidation of methyleneto OH, CO₂R, or OR, oxidation of arylmethanes, oxidation of ethers tocarboxylic esters and related reactions, oxidation of aromatichydrocarbons to quinones, oxidation of amines or nitro compounds toaldehydes, ketones, or dihalides, oxidation of primary alcohols tocarboxylic acids or carboxylic esters, oxidation of alkenes to aldehydesor ketones, oxidation of amines to nitroso compounds and hydroxylamines,oxidation of primary amines, oximes, azides, isocyanates, or nitrosocompounds, to nitro compounds, oxidation of thiols and other sulfurcompounds to sulfonic acids), reactions in which oxygen is added to thesubstrate (e.g., oxidation of alkynes to alpha-diketones, oxidation oftertiary amines to amine oxides, oxidation of thioesters to sulfoxidesand sulfones, and oxidation of carboxylic acids to peroxy acids, andoxidative coupling reactions (e.g., coupling involving carbanoins,dimerization of silyl enol ethers or of lithium enolates, and oxidationof thiols to disulfides).

Exemplary reductive reactions include, for example, reactions involvingreplacement of oxygen by hydrogen {e.g., reduction of carbonyl tomethylene in aldehydes and ketones, reduction of carboxylic acids toalcohols, reduction of amides to amines, reduction of carboxylic estersto ethers, reduction of cyclic anhydrides to lactones and acidderivatives to alcohols, reduction of carboxylic esters to alcohols,reduction of carboxylic acids and esters to alkanes, complete reductionof epoxides, reduction of nitro compounds to amines, reduction of nitrocompounds to hydroxylamines, reduction of nitroso compounds andhydroxylamines to amines, reduction of oximes to primary amines oraziridines, reduction of azides to primary amines, reduction of nitrogencompounds, and reduction of sulfonyl halides and sulfonic acids tothiols), removal of oxygen from the substrate {e.g., reduction of amineoxides and azoxy compounds, reduction of sulfoxides and sulfones,reduction of hydroperoxides and peroxides, and reduction of aliphaticnitro compounds to oximes or nitriles), reductions that include cleavage{e.g., de-alkylation of amines and amides, reduction of azo, azoxy, andhydrazo compounds to amines, and reduction of disulfides to thiols),reductive coupling reactions {e.g., bimolecular reduction of aldehydesand ketones to 1,2-diols, bimolecular reduction of aldehydes or ketonesto alkenes, acyloin ester condensation, reduction of nitro to azoxycompounds, and reduction of nitro to azo compounds), and, reductions inwhich an organic substrate is both oxidized and reduced {e.g., theCannizzaro reaction, the Tishchenko reaction, the Pummererrearrangement, and the Willgerodt reaction).

Examples of cleavable linkers/protecting groups that may be cleaved inorder to transfer molecule fragments as described above, or may be usedto protect and de-protect functional groups during the synthesis of thelibrary of bi-functional molecules, and conditions mediating thecleavage of these linkers/protecting groups, are shown in FIG. 8.Cleavable linkers can be cleaved in any number of ways, e.g., byphotolysis or increased temperature, or by the addition of acid, base,enzymes, ribozymes, other catalysts, or any other agents.

The linkers of the present invention may for example be chosen from thefollowing list: Carbohydrates and substituted carbohydrates, polyvinyl,acetylene or polyacetylene, aryl/hetaryl and substituted aryl/hetaryl,ethers and polyethers such as e.g. polyethylenglycol and substitutedpolyethers, amines, polyamines and substituted polyamines, single- ordouble-stranded oligonucleotides, and polyamides and natural andunnatural polypeptides.

To maintain a physical link between the identifier and the encodedmolecule (in the case of stage 2 synthesis, the template and the encodedmolecule), at least one non-cleavable linker is needed. Thenon-cleavable linker may of course be cleavable under certainconditions, but is non-cleavable under the conditions that lead to thebi-functional molecule employed in the screening. This non-cleavablelinker is preferably flexible, enabling it to expose the encodedmolecule in an optimal way. Preferably the length of the flexible linkeris in the range of 1-50 Å, more preferably 5-30 Å, most preferably 10-25Å. Preferably the linker is both flexible and inert; polyethylene glycol(PEG) is an appropriate linker.

Under certain conditions it may desirable to be able to cleave thelinker after the screening of the library of bi-functional molecules hasbeen done, for example in order to perform a mass spectrometric analysisof the encoded molecule without the identifier attached, or to performother types of assays on the free encoded molecule.

In an alternative embodiment, the linker contains an oligonucleotidemoiety which may serve as an annealing site for an oligonucleotide thatcarries a reagent, catalyst or molecule fragment. The annealing of thereagent-, catalyst- or molecule fragment-oligonucleotide will serve toprovide the reagent, catalyst or molecule fragment in a high localconcentration, thereby improving the efficiency of the desired reaction.

Alternative Methods for Templated Synthesis Using Bi-Functional CarrierMolecules:

Alternative methods (to the example shown in FIG. 2) can be envisionedfor carrying out the template directed reactions set in stage 2 of theinvention. The alternative methods provide different ways of bringingtogether and reacting the bi-functional carrier molecules provided bystage 1. Some examples of such alternative methods are shown in FIG. 4.Any of these methods could be used to carry out stage 2 of theinvention.

Example 1 See Example 1, FIG. 4

This is identical to the example provided in FIG. 2, except that thecarriers are ligated together before reaction. X and Y represent the twodifferent repertories of carrier molecules from stage 1 used in theexample of FIG. 2. Once the oligonucleotide identifiers of the carriershave annealed to the template, they are ligated to each other and theacyl transfer reaction is hereafter carried out (the acyl transfer couldalso be carried out prior to the ligation step). The ligation step maybe performed by a ligase, or may be performed in absence of an enzyme.

Example 2 See Example 2, FIG. 4

In example 2, the molecule fragments are attached to the ends of theoligonucleotide identifiers that are distal to each other afterannealing to the template. After ligation of the oligonucleotideidentifiers of the carriers, the molecule fragments are brought incontact for reaction by denaturing the duplex DNA (ligated carriersannealed to template) and removal of the template (in order to haveincreased flexibility of the now single stranded DNA sequence thatcarries the molecule fragments). The correct juxtaposition of themolecule fragments can be further ensured by including complementarysequences in the ends of the oligonucleotide identifiers that areproximal to the molecule fragments before transfer.

After reaction of the molecule fragments the single stranded DNA can bemade double stranded by a standard extension reaction using a DNApolymerase and a primer annealing to the end of the DNA.

Example 3 See Example 3, FIG. 4

Example 3 shows how the oligonucleotide identifier of the carrier thatcarries the combined molecule fragments can be ligated to the template.This is achieved by including a region of self-complementary sequence onthe template that after annealing will juxtapose the ends of the carrieroligo and the template.

As in example 2 it may also in this example be desirable to perform anextension reaction, using a primer that anneals to one of the ends ofthe DNA strand that is attached to the encoded molecule and a DNApolymerase. The resulting double stranded DNA, where the encodedmolecule is displayed at the end, is shown in the figure of the example.

Example 4 See Example 4, FIG. 4

In this example of the reaction in stage 2 of the invention, thecarriers employed carry double-stranded oligonucleotide identifiers(generated for example by ligation of double-stranded oligonucleotideidentifiers during the Stage 1 synthesis). Thus, these carriers arebrought together and reacted without the use of a template. Theoligonucleotides of the carrier are constructed so that the resultingdouble stranded DNA of one repertoire of carriers (e.g. X) has a“sticky” end (a 3′ or 5′ overhang), which is complementary to acorresponding “sticky” end of another repertoire of carriers (e.g. Y).Any two carrier molecules from each repertoire can therefore be combinedby annealing using the sticky ends of the double stranded DNA oligos.The carriers are hereafter ligated and reacted like in the otherexamples. The identifier DNA sequence of the combined molecules will, inaddition to the 48-mer coding sequence, contain a “sticky” end derivedsequence that will be identical for all molecules. One advantage of thismethod is that synthesis of long templates is avoided.

After reaction of the molecule fragments the single stranded DNA can bemade double stranded by a standard extension reaction using a DNApolymerase and a primer annealing to the end of the DNA.

In examples 1-4 it may be desirable to include primer binding sites,either at the ends of the template DNA strand, or as part of theidentifier (one primer binding site per carrier) when stage 2 involves aligation of two carriers to each other, to form a complementarytemplate. These primer binding sites can be used for amplification ofthe template (or complementary strand), allowing generation of morecopies of the encoded molecules.

Example 5 See Example 5, FIG. 4

Carrier molecules with double-stranded oligonucleotide identifiers, asprovided in example 4, can also be ligated into a circular doublestranded DNA molecule, by digestion of the double stranded DNA moleculewith restriction enzymes, which creates “sticky ends”, and ligation tocorresponding “sticky ends” on the double-stranded oligonucleotideidentifiers. The double stranded DNA molecule used for ligation of theoligonucleotide identifiers can be a plasmid having a replicationorigin, which enables it to be transformed into cells, e.g. bacteria,for amplification. The double stranded DNA molecule can also be anon-functional piece of DNA only used to make circular DNA moleculescontaining the double-stranded oligonucleotide identifiers.

In the first step the carrier molecules from one repertoire of synthesisin stage 1 are ligated into the circular DNA. In the second step, thecarrier molecules from another repertoire of synthesis in stage 1 areligated into the double-stranded DNA molecule, adjacent to where thecarrier molecules from the first step were inserted. The carriermolecules are hereafter reacted as in the other examples.

Synthesis of the Template:

10¹² different templates are required in order to generate a library of10¹² encoded molecules. In the example shown in FIGS. 1 and 2, thelibrary consists of tetrameric polypeptides that are encoded by a 48-mertemplate sequence. Each of the four amino acid units in an encodedmolecule is encoded by a 12-mer sequence-unit of the DNA template. Inthe examples, the number of different amino acids that are encoded bythe 12-mer sequence-units is 1000. A simple and efficient way ofsynthesising the 10¹² DNA templates in the example is thus bysynthesising the 1000 different 12-mer oligonucleotides, correspondingto the first position of the template. This set of position 1oligonucleotides are then incubated with a corresponding set of position2, 3 and 4 oligonucleotides, and ligated, to form templates comprisingfour 12-nucleotide sequence units. A practical approach to this issuggested in FIG. 5:

i) Four sets of double-stranded DNA fragments are generated, withoverhangs at both ends. The overhangs may be of any length, for exampleone-nucleotide overhangs. Thus, for each of the four sets, each of 1000different 13-nt DNA oligo sequences are incubated with a partiallycomplementary 13-nt DNA oligo. 12 of 13 nucleotides are complementary,and therefore duplex DNA with one-nucleotide overhangs at both ends isgenerated.ii) Then the 4 sets each of 1000 duplex DNA fragments are incubated, andligated together by a DNA ligase to form 10¹² different DNA templates,each comprising four sequence-units of 12 base pairs. A set of primerannealing sequences can subsequently be ligated to the ends of thetemplates, in order to obtain templates that can easily be amplified byPCR using primers complementary to the flanking sequences.Alternatively, as shown in FIG. 5, the primer-annealing-sites may becarried by the terminal sequence units (in this example comprisingposition 1 and position 4 sequence-units). The primer binding sites alsoallow sequencing of the templates.iii) Single-stranded template is generated by removing the upper orlower strand, for example by including biotinylated position 1 upperstrand oligos. Streptavidin beads may be added, which will immobilisethe double stranded templates on the beads. Then alkaline conditions areemployed to melt the DNA duplexes, which releases the lower strand fromsolid support. The single stranded DNA templates may now be used in theStage 2 DNA-templated synthesis reactions. Alternatively, theDNA-templated synthesis may be performed using the immobilised upperstrand as template, and thus performing the reactions on solid support.This should allow the DNA-templated reactions to be performed underaqueous conditions as well as in organic solvents.

The library of DNA templates may be generated in other ways. Forexample, 10¹² different templates may be generated by standardoligonucleotide synthesis of fully random oligonucleotides, oroligonucleotides with constant, partly randomised, and/or fullyrandomised positions. Alternatively, the template may be generated bysplit and mix synthesis, as described below.

The Nucleic Acid Component of Bi-Functional Molecules Generated in Stage1 and Stage 2 Synthesis.

The present invention involves oligonucleotide identifiers of threedifferent uses. Thus, “unit identifier” is the smallest unit, andtypically describes the part of the final encoded molecule that is addedand becomes attached to the nascent bi-functional molecule as the resultof a synthesis round during stage 1 synthesis. “Carrier identifier”describe the oligonucleotide that anneals to the template during a stage2 synthesis round. Finally, “template identifier” or “identifiertemplate” describes the encoding oligonucleotide of the bi-functionalmolecule. Thus, in the example described in FIGS. 1 and 2, there are 4unit identifiers that are linked two and two to generate 2 carrieridentifiers. Then, the two carrier identifiers are hybridised to 1identifier template carrying four identifiers complementary to theidentifier portions of the carriers. In this example, the encodedmolecule is attached to the identifier template non-covalently byannealing of the oligonucleotide that carries the encoded molecule tothe identifier template.

In most of the examples, two molecule fragments and two unit identifiersare used to generate a carrier molecule, but the number of moleculefragments and unit identifiers used to synthesise a carrier can also be1, 3, 4, 5, 6, 7 or higher. The resulting molecules are termed carriermolecules, where a given carrier molecule contains molecule fragmentsthat are linked together, and where the linked molecule fragments arealso linked to the identifiers that have also been linked together.

The number of carriers that bind to the same template may be 1, 2, 3, 4,5, 6, 7 or more.

The identifiers of carrier molecules must be capable of hybridising tothe template in a sequence- or partly sequence-specific way. Thus, in apreferred embodiment the template is a nucleic acid or nucleic acidanalogue, and the identifiers of the carrier molecules consist of DNA,RNA, PNA, LNA, or other oligonucleotide analogues capable of sequencespecific hybridisation through base pairing. The resulting structure maybe a double or triple helix.

The templates employed during stage 2 synthesis likewise must be capableof hybridising to the carriers in a sequence-specific or partlysequence-specific way. Thus, the templates preferably consist of DNA,RNA, PNA, LNA, or other oligonucleotide analogues capable of sequencespecific hybridisation through base pairing. In a preferred embodiment,the template is amplifiable (i.e. can be amplified through the use of apolymerase, such as is used in a PCR-reaction, or by repeatedreplication such as in a cell). Example amplifiable templates are DNAand RNA, and unnatural DNA- or RNA, capable of being used as a templatein a polymerase-based transcription or replication process.

The carrier identifiers preferably anneal sequence specifically to thetemplates. Therefore, the carrier identifiers should be of a length andcomposition that allows a relatively strong and specific interactionbetween template and carrier under the hybridisation step. Preferably,the length of the carrier identifier is in the range of 3-50nucleotides, more preferably 7-25 nucleotides, and most preferably 8-20nucleotides. In order to ensure specific annealing of the carriers, i.e.to ensure that the correct carrier anneal to a given template, the setof carrier identifiers should be chosen such that the overlap betweenidentifiers of different carriers is as insignificant as possible. Inother words, the design of the set of carrier identifiers should ensurea high degree of non-identity among any carrier identifier in the set.Likewise, the templates should be designed so that interaction withundesired carrier identifiers is minimised.

Preferably, less than 30% of the nucleotides of a given carrieridentifier sequence should be complementary to a non-desiredhybridisation sequence of the template. Less preferably, less than 50%,and even less preferably, less than 70%, and the least preferably, lessthan 90%, of the nucleotides of a given carrier identifier sequenceshould be complementary to a non-desired hybridisation sequence of thetemplate. This will ensure a high degree of specificity during theannealing step in the templated synthesis, and hence, ensure that thedesired carriers bind to the template, and hence, ensure that thedesired encoded molecules are generated. In principle, even where twocarrier identifiers are different only at one nucleotide position, thestage 1 and stage 2 synthesis and encoding/decoding should work.However, the more different any two identifiers in a library are, themore robust the encoding, selection and characterisation will be.

In addition, the more different the identifier template sequences are,the less sensitive the system will be to errors introduced during thechemical reactions or PCR amplifications performed. For example, if allunit identifier pairs have different nucleotides at more than 5positions, two nucleotide substitutions introduced during PCR will stillallow the correct identification of the unit identifier. Therefore, thenon-identity preferences that are described above for carrieridentifiers also applies to unit identifiers employed during stage 1synthesis, also if no stage 2 synthesis is performed to generate thebi-functional molecules employed in the screening step.

In order to obtain efficient attachment of the incoming identifier andthe nascent bi-functional molecule during stage 1 synthesis, it ispreferable to use oligonucleotide identifiers that have constantregions, i.e. a portion of the identifier is the same for all of theidentifiers in the library. In the stage 1 synthesis, this region couldbe relatively long (e.g., 20 nucleotides), to ensure efficient annealingto a complementary oligonucleotide, and hence, efficient ligation.However, if the bi-functional molecule produced in this way is to beused as a carrier molecule in a subsequent stage 2 synthesis, theconstant regions should be as short as possible, in order to ensure highannealing specificity among the different carrier molecules being usedin the stage 2 synthesis. An appropriate compromise between these twoopposing factors is to include constant regions (for mediating theligation of unit identifiers) that are preferably between 1 and 10nucleotides long, less preferably 1-20 and least preferably 1-50nucleotides long. Most preferably, the constant region is 2, 3, 4, 5, or6 nucleotides long. In order to ensure efficient annealing and thusligation, but to reduce the length of the constant region, the constantregion may include unnatural oligonucleotides such as e.g. LNA, whichprovides a higher affinity then natural DNA.

Variations and Specifications to the General Scheme Described Above forthe Generation of Bi-Functional Molecules:

A number of methods by which to generate carriers exist. In principle,any number of methods exist by which to perform the templated reactions.

Almost any combination of methods for carrier generation and templatedsynthesis can be applied to the generation of bi-functional molecules.Below specific approaches to carrier generation have been outlined(Sub-procedures 1-X) and specific approaches to templated synthesis havebeen outlined (Sub-processes A-Z). Individual processes 1-X and A-Z, aswell as combinations 1A, 1B, . . . 1Z, 2A, 2B, . . . , XA, XB, . . . ,XZ can be applied to the generation of bi-functional molecules.

Specific enablements of stage 1 synthesis, or generation of carriers,include but is not limited to:

This is a variation of bi-functional molecule formation described in(Lerner et al., EP 0643778 B1, Encoded combinatorial chemicallibraries), but any embodiment of bi-functional molecule synthesisdescribed in said patent is applicable for stage 1 synthesis. Twoalternating parallel combinatorial syntheses are performed so that agenetic tag is chemically linked to a polypeptide or other type oforganic molecule being synthesised; in each case, the addition of oneamino acid residue (or other type of molecule fragment with at least onereactive group) to the structure is followed by the addition of anoligonucleotide sequence, which is defined to code for that amino acid(or molecule fragment), i.e., to function as an identifier for thestructure of the amino acid residue (or molecule fragment). The libraryis built up by the repetition of this process after pooling anddivision. During the process, therefore, a bi-functional molecule isbeing formed, consisting of a polypeptide (or other organic molecule)attached to an identifier tag that encodes the synthetic history of theencoded molecule, and hence, encodes the expected chemical structure ofthe encoded molecule. Thus, the process is an example of a split-and-mixDNA tagging process.

In a related process, also described in (Lerner et al., EP 0643778 B1,Encoded combinatorial chemical libraries), a method is described forpreparing tagged molecules or libraries of tagged molecules, where eachmolecule is attached to a solid support. The method comprises a solidsupport, being dispersible in aqueous solution, a first linkage unitcoupled to the solid support, a second linkage unit coupled to the firstlinkage unit, a bi-functional unit coupled to the second linkage unit,wherein the bi-functional unit (or “linker”) has two reactive groups,one of which is employable for oligonucleotide synthesis (i.e., may beused as an initiator-functionality for oligonucleotide synthesis), andone of which is employable for polypeptide- (or other organic molecule-)synthesis.

This is a variation of bi-functional molecule formation described in(Dower et al., EP 0604552 B1), but any embodiment of bi-functionalmolecule synthesis described in said patent is applicable for stage 1synthesis. Organic molecules are synthesized in a component by componentfashion (i.e. by a split-and-mix-like process) on solid support orparticles. During the synthesis of the organic molecule, a tag issimultaneously synthesised which becomes linked to the organic moleculevia a linker. The organic molecule-oligonucleotide tag complex may bereleased from these supports to provide a soluble library ofbi-functional molecules.

This is a variation of bi-functional molecule formation described in(Freskgård et al., WO 2004/039825 A2), but any embodiment ofbi-functional molecule synthesis described in said patent application isapplicable for stage 1 synthesis. A bi-functional carrier molecule isgenerated as follows: A nascent bi-functional complex comprising areactive group and a priming site for enzymatic addition of anoligonucleotide identifier is reacted at the reactive group with one ormore molecule fragments, and provided with respective identifier(s)identifying the molecule fragments at the priming site using one or moreenzymes. The enzyme used may be a DNA ligase, and the priming site maybe a 3′-OH group on a nucleic acid to which an oligonucleotide tagcarrying a 5′-phosphate can be attached. In a preferred embodiment, theligation of identifiers does not involve an enzyme. Enzymes, includingthe DNA ligase, are in general substrate specific, entailing that theenzymatic addition of a tag to the priming site is not likely tointerfere with the display molecule being formed. Generally, thebi-functional carrier molecule is formed by more than a single round ofreaction between one or more molecule fragments and the reactive group.In a certain aspect of the invention, the nascent bi-functional complexreacted with one or more molecule fragment(s) and provided withrespective oligonucleotide identifier(s) is reacted further one or moretimes with one or more molecule fragment(s) and is provided withrespective identifier(s) to produce an encoded molecule as one part ofthe bi-functional carrier molecule and an encoding part comprisingidentifiers which codes for the identity of the molecule fragments whichhave participated in the formation of the encoded molecule. The reactionat the reactive group and the addition of identifiers may occur in anyorder, i.e. the reaction may occur subsequent to, simultaneously with,or previous to the identifier addition. The choice of order may amongother things be dependent on the enzyme type, the reaction conditions,and the type of reactant. The encoding part of the nascent bi-functionalcomplex is formed by addition of at least one unit identifier to apriming site using one or more enzymes. Further unit identifiers may beattached to a previous unit identifier so as to produce a linear orbranched encoding part. As long as at least one unit identifier isattached by an enzymatic catalysed reaction, further unit identifiersmay be provided using chemical means or enzymatic means at thediscretion of the experimenter. The identifier can be added to thepriming site using any appropriate enzyme. In a certain embodiment, anidentifier is provided at the priming site of the nascent bi-functionalcomplex utilizing an enzymatic extension reaction. The extensionreaction may be performed by a polymerase or a ligase or a combinationthereof. The extension using a polymerase is suitably conducted using acomplementary oligonucleotide that hybridises to the previous unitidentifier, and carries a sequence complementary to the unit identifier;the unit identifier is then synthesized by e.g. a polymerase by usingthe complementary oligonucleotide as template, and the end of theprevious unit identifier as starting point for the extension. Typically,the previous unit identifier would carry a 3′-OH at the end, and apolymerase would extend from this 3′-OH, using dNTPs or NTPs. Examplesof suitable enzymes for the addition of unit identifiers to the nascentbi-functional molecule include DNA polymerase, RNA polymerase, ReverseTranscriptase, DNA ligase, RNA ligase, Taq DNA polymerase, Pfupolymerase, Vent polymerase, HIV-1 Reverse Transcriptase, Klenowfragment, or any other enzyme that will catalyze the incorporation ofcomplementing elements such as mono-, di- or polynucleotides.Polymerases that allow mismatch extension can also be used, such as forexample DNA polymerase r\ (Washington et al., (2001) JBC 276:2263-2266), DNA polymerase i (Vaisman et al., (2001) JBC 276:30615-30622), or any other enzyme that allow extension of mismatchedannealed base pairs. In another aspect, when ligases are used, suitableexamples include Taq DNA ligase, T4 DNA ligase, T4 RNA ligase, T7 DNAligase, and E. coli DNA ligase. The choice of the ligase depends to acertain degree on the design of the ends to be joined together. Thus, ifthe ends are blunt, T4 RNA ligase may be preferred, while a Taq DNAligase may be preferred for a sticky end ligation.

This is another variation of bi-functional molecule formation describedin (Freskgård et al., WO 2004/039825 A2). In this approach, moleculefragments are attached to the unit identifier or an oligonucleotide thatis complementary to the unit identifier. Using this approach, thesynthesis of a library of many different bi-functional carrier moleculescan be conducted in a single vessel, in contrast to the split-and-mixsynthesis, where the reaction and unit identifier addition must becarried out in separate compartments. Thus, this variation entails amethod comprising the steps of i) providing a nascent bi-functionalcomplex comprising a reactive group and an oligonucleotide identifierregion, ii) providing a molecule fragment-oligonucleotide conjugatecomprising an oligonucleotide sufficient complementary to theoligonucleotide identifier to allow for hybridisation, a transferablemolecule fragment, and a complementary anti-codon identifying thefunctional entity, iii) mixing the nascent bi-functional molecule andthe molecule fragment-oligonucleotide conjugate under hybridisationconditions to form a hybridisation product, iv) transferring themolecule fragment of said conjugate to the nascent bi-functionalmolecule through a reaction involving the reactive group of the nascentbi-functional complex, and v) enzymatically extending theoligonucleotide identifier to obtain a unit identifier attached to thebi-functional molecule having received the molecule fragment. Theenzymatic extension may occur subsequent to or simultaneously with thetransfer of the functional entity or even prior to the transfer.

This is a variation of bi-functional molecule formation described in(Lerner et al., EP 0643778 B1, “Encoded combinatorial chemicallibraries” and Dower et al., EP 0604552 B1). In this method, a stage 1synthesis is performed in which identifiers are ligated together in theabsence of an enzyme, but with the aid of a nucleic acid that brings theidentifiers into close proximity, and hence increases ligationefficiency this way (see FIG. 12). An identifier that must be ligatedmust comprise, e.g. at one end of the identifier oligonucleotide, achemical group that can react with a chemical group on the identifierthat it will be ligated to. As an example, the nucleic acid may becomplementary to a part of the sequence of both identifiers. In apreferred embodiment a nucleic acid is added that is complementary tothe ends of the identifiers. Hybridisation of this bridging nucleic acidto the two identifiers brings the two chemical groups, one from eachidentifier, into close proximity, thereby improving the ligationreaction efficiency, or nucleic acid analog, or any other type ofmolecule or solid support that brings the two identifiers into proximityand thereby increases the efficiency of ligation.

The complementary nucleic acid brings the ends of the two identifiersinto close proximity; the ends of the identifiers have been modified tomake them prone to reaction under these conditions. As an example ofsuch chemical ligation, one of the identifiers may carry a 3′-hydroxylgroup, and the other identifier carry a 2-methylimidazole-activatedphosphate at its 5′-end. When brought into proximity by hybridisation toa complementary nucleic acid, the 3′-OH and the 5′ activated phosphatewill react to generate a native phosphodiester bond between theidentifiers. This and other types of chemical ligation reactions thatcan be used to ligate identifiers together chemically are described inmore detail above. Practically, any two reactive groups that may reactto form a bond between the two identifiers can be used, as long as theligation conditions do not modify the identifiers to an extent thatabolishes efficient hybridisation of the carriers in the stage 2synthesis that follows carrier synthesis. Thus, as an example, thereactive groups and the reactions listed in FIGS. 6 and 7 may be usedfor ligation of identifiers as well.

The encoded molecule formed using this approach, and the reactive groupsand types of reactions that may be used to generate the encodedmolecule, are the same as those mentioned for general stage 1 synthesis(and stage 2 synthesis), and thus include reactions and reactive groupslisted in FIGS. 6, 7 and 8.

This variation of combinatorial chemistry combines the combinatorialchemical synthesis of molecule fragments with a tagging of each moleculesynthesized. Combinatorial chemistry is employed to synthesize a libraryof molecule fragments, whereafter the molecule fragments are attached tooligonucleotides, to produce bi-functional molecules, that may be usedas carriers in templated synthesis. In this approach, combinatorialchemistry is applied to the generation of a library of moleculefragments. Any type of combinatorial chemistry may be used, for examplesplit-and-mix synthesis on beads (see review by Abelson (1996) Methodsin Enzymology, vol. 267, p. 211-221; Lebl et al. (2000) U.S. Pat. No.6,090,912; Lebl et al. (1998) U.S. Pat. No. 5,840,485), followed by therelease from the bead to produce multiple solutions (e.g. in separatewells), each containing a specific molecule fragment, or the library maybe generated in an array format, for example on glass or pins (Dapremontet al. (1995) Physiol. Chem. Phys. & Med. NMR, 27: 339-343, “Multiplesynthesis using the multipin method”), before it is eventually releasedfrom solid support, to produce multiple solutions (e.g. in separatewells), where each well contain a specific molecule fragment. Any othermeans of generating a library of compounds can be applied, including theembodiments of (Still et al. (1998) U.S. Pat. No. 5,721,099; Dower etal. (1991) US 1991000762522; Boger et al. (2001) U.S. Pat. No. 6,194,612B1; Cook et al. (2001) U.S. Pat. No. 6,191,273; Gustafson et al. (2000)U.S. Pat. No. 6,140,361; Graybill et al. (2000) U.S. Pat. No. 6,127,191;Dervan et al. (2000) U.S. Pat. No. 6,090,947; Baindur et al. (1999) U.S.Pat. No. 5,891,737; Baindur et al. (1997) U.S. Pat. No. 5,646,285). Oncethe library of molecule fragments is generated, each specific moleculefragment is attached a specific oligonucleotide. As a result, a libraryof bi-functional molecules have been generated, where specific moleculefragments are attached to specific oligonucleotides that thus encode thestructure of the molecule fragment generated by combinatorial chemistry.The bi-functional molecules may be used as carriers in a templatedsynthesis.

In a preferred embodiment, the oligonucleotides are prepared bycombinatorial synthesis, either before or after their addition to thesolutions containing the specific molecule fragments. Thus, for thetagging of n² molecule fragments, n oligonucleotides of length 12 nt arepairwise attached, for example by chemical ligation, to noligonucleotides of length 13 nt, to produce n² oligonucleotides oflength 25 nt that is then attached to the n₂ molecule fragments, forexample by an acylation reaction involving an amino group on one of theends of the oligonucleotides, and a carboxylic acid of the moleculefragment. For example, if a library of 10⁶ different molecule fragmentshave been prepared, one may generate 10⁶ different oligonucleotides bypairwise ligation of two sets of 10³ oligonucleotides.

This is a variation of the principle described immediately above, wherecombinatorial chemical synthesis is replaced by any synthetic methodthat produces a library of more than one hundred, one thousand, or tenthousand molecule fragments. In this variation it is thus not arequirement that a combinatorial approach is employed for the synthesisof the library of molecule fragments. Again, the bi-functional moleculesare generated by attachment of specific oligonucleotides to each of themolecule fragments, and again, the oligonucleotides can be attached tothe molecule fragments directly, or after a combinatorial ligation of asmaller set of oligonucleotides has produced a larger set ofoligonucleotides that may then be attached to the molecule fragments.Again, the combinatorial ligation of oligonucleotides can also beperformed after attachment of the first oligonucleotide to the moleculefragment.

This is a variation of any of the other stage 1 synthesis principles, orformation of carrier molecules (1-8), in which identifiers are linked bya non-covalent bond. As an example, the identifiers may containcomplementary oligonucleotide sequences. In a preferred embodiment, astrong, non-covalent association of the identifiers by extendedcomplementarity may allow hybridisation of the hybridised identifiers(the carrier molecule) to a template in a subsequent templatedsynthesis.

This is a variation of Morgan et al., 2005, WO 2005/058479. Anyembodiment of bi-functional molecule synthesis described in said patentapplication is applicable for stage 1 synthesis The invention provides amethod of synthesising libraries of molecules which include an encodingoligonucleotide tag. The method utilises a “split and pool” strategy inwhich a solution comprising an initiator (similar to the linkermolecules described in this patent application), comprising a firstbuilding block linked to an encoding oligonucleotide, is divided intomultiple fractions. In each fraction, the initiator is reacted with asecond, unique, building block and a second, unique oligonucleotidewhich identifies the second building block (the oligonucleotides arereacted through the use of an enzyme, e.g. a ligase). These reactionscan be simultaneous or sequential and, if sequential, either reactioncan precede the other. The dimeric molecules produced in each of thefractions are combined and then divided again into multiple fractions.Each of these fractions is then reacted with a third unique buildingblock and a third unique oligonucleotide which encodes the buildingblock (the oligonucleotides are reacted through the use of an enzyme).The number of unique molecules present in the product library is afunction of the number of different building blocks used at each step ofthe synthesis, and the number of times the pooling and dividing processis repeated.

This is a variation of Harbury and Halpin (WO 00/23458). Any embodimentof bi-functional molecule synthesis described in said patent applicationis applicable for the synthesis of bi-functional carrier molecules. Themethod involves the synthesis of a plurality of compounds, comprising a)forming a first group of subsets of identifier templates, where thetemplates in each subset each has a selected one of a plurality ofdifferent first hybridisation sequences, a mixture of different secondhybridisation sequences, and a reactive group, b) reacting the reactivegroup in each of the subsets formed in (a) with a selected moleculefragment, thereby to form a molecule fragment-specific compoundintermediate on the associated sequence in each subset, c) forming asecond group of subsets of the reacted templates, where the templates ineach subset each have a selected on of a plurality of a plurality ofdifferent second hybridisation sequences, and a mixture of differentfirst hybridisation sequences; and d) reacting the compoundintermediates in the sequences in each of the subsets formed in (c) witha selected molecule fragment. The method may be performed on solidsupport, for example by sorting the templates according to the sequenceof e.g. the first hybridisation sequence by annealing to beads, each ofwhich carry a specific oligonucleotide that is complementary to aspecific first hybridisation sequence, to allow the formation ofdifferent subsets of templates, where each subset of templates have aspecific hybridisation sequence. The method thus provides subsets ofnucleic acid templates, generated by by base-specific duplex formationbetween each different first hybridisation sequence and a complementaryoligonucleotide. The reactive group in each of the subsets are reactedwith a selected molecule fragment to form a molecule fragment-specificcompound intermediate (i.e., a bi-functional molecule comprising aspecific molecule fragment). The end result thus is, after grouping thetemplates into subsets each comprising a particular first hybridisationsequence, and reacting each subset with a specific molecule fragment,and repeating this process for the second, third, fourth, etc.hybridisation sequence, a number of bi-functional molecules, comprisingan encoded molecule (comprising n linked molecule fragments) and anencoding template identifier, comprising n hybridisation sequences (herecalled unit identifiers).

The templates employed can carry 1, 2, 3, 4, 5, 6, 7 or morehybridisation sequences, and thus, the bi-functional molecules generatedby this approach can carry 1, 2, 3, 4, 5, 6, 7 or more moleculefragments. The number of different molecule fragments in a given roundcan be from 2 to 10.000. The template may be amplifiable ornon-amplifiable by polymerases such as Taq polymerase

The method, as described above, as well as the embodiments described in(Harbury and Halpin, WO 00/23458) may be performed using the chemistriesand protecting groups described in this patent application, includingchemistries and protecting groups shown in table 6, 7 and 8, and usingany type, composition and length of oligonucleotides described in thispatent application.

Specific enablements of stage 2 synthesis (templated synthesis ofbi-functional molecules) include but is not limited to:

This is a variation of the principle described in (Bruick et al.,Chemistry and Biology. January 1996, 3: 49-56, “Template-directedligation of peptides to oligonucleotides”). In this approach a number ofidentifier templates are incubated with two (or more) sets of carriermolecules. One set of carriers comprise a molecule fragment comprising areactive group A (for example an activated ester of a carboxylic acid,for example a thioester), and one set of carriers comprise a reactivegroup B that can react with A (B can be for example a primary orsecondary amine, capable of reacting with the thioester). Upontemplate/carrier complex formation, an acylation reaction takes place,leading to the transfer of the molecule fragment comprising the reactivegroup A (e.g. thioester) onto the molecule fragment comprising thereactive group B (e.g. the amine). Thus, the templated reaction leads toformation of an encoded molecule consisting of two molecule fragments,linked by an amide bond.

Any type of reactive groups may be used in this method, includingreactive groups mentioned in this patent application, including table 6,7, and 8. For amine acylations, thioesters, N-hydroxysuccinimide esters,and phenol esters are particularly well suited for the direct transferof one molecule fragment onto another. Alternatively, an indirectacylation between a carboxylic acid and an amine may be mediated by EDC,EDC/NHS, DMT/MM and other reagents that activate the acid fornucleophilic attack. After covalent linkage of the two moleculefragments, one of the linkers is cleaved, to transfer one moleculefragment onto the other molecule fragment, associated with the sametemplate. In addition, many other reactions may be used to link twomolecule fragments in an oligonucleotide-templated fashion. Thus, thisprinciple may be applied to the generation of encoded molecules aslisted in the present invention, and may involve reactive groupsmentioned in the present invention, including those listed in FIGS. 6,7, and 8.

This is a variation of the principle described in (Walder et al. (1979)Proc. Natl. Acad. Sci. USA, 76: p. 51-55), and also is closely relatedto the principle described immediately above. In this approach, theformation of an amide bond between two molecule fragments is facilitatedby the juxtaposition of these by hybridisation of two oligonucleotides,each carrying one molecule fragment, to a complementary template. Anoligonucleotide, in the present invention termed a carrier identifier,to which a carboxylic acid of a molecule fragment is attached throughester linkage, is hybridised to a template, which is also hybridised toa second oligonucleotide carrying a molecule fragment comprising anamino group. The duplex DNA is designed so as to bring the ester and theamine into close proximity. Because of this proximity, a reactionbetween the ester and the amine takes place; this leads to formation ofan amide bond between the two molecule fragments, and the ester iscleaved to allow transfer of the ester-bound molecule fragment onto themolecule fragment comprising the amino group. An ester linkage is thusused to allow direct transfer of the molecule fragment; in principle anysubstitution-labile acyl linkage should allow the direct transfer of onemolecule fragment onto the other in this scheme. In addition, many otherreactions may be used to link two molecule fragments in anoligonucleotide-templated fashion. Thus, this principle may be appliedto the generation of encoded molecules as listed in the presentinvention, and may involve reactive groups mentioned in the presentinvention, including those listed in FIGS. 6, 7, and 8.

This is a variation of (Liu et al. (2002), WO 02/074929 A2, “Evolvingnew molecular function”). Any embodiment of bi-functional moleculesynthesis described in said patent application is applicable for thestage 2 synthesis of bi-functional molecules. In a preferred embodimentof this application, the method comprises first providing one or morenucleic acid templates, which one or more nucleic acid templatesoptionally have a reactive unit associated therewith. The nucleic acidtemplate is then contacted with one or more transfer units designed tohave a first moiety, an anticodon, which hybridises to a sequence of thenucleic acid, and is associated with a second moiety, a reactive unit,which includes a building block of the compound to be synthesised. Oncethese transfer units have hybridised to the nucleic acid template in asequence-specific manner, the synthesis of the chemical compound cantake place due to the interaction of reactive moieties present on thetransfer units and/or the nucleic acid template. Significantly, thesequence of the nucleic acid can later be determined to decode thesynthetic history of the attached compound and thereby its structure.The method allows the synthesis of large numbers of molecules usingcombinatorial methods. The principle may be applied to the generation ofencoded molecules as listed in the present invention, and may involvereactive groups mentioned in the present invention, including thoselisted in FIGS. 6, 7, and 8.

D. This is a variation of the templated synthesis described in (Pedersenet al. (2002) WO 02/103008 A2, “Ternplated molecules and methods forusing such molecules”). Any embodiment of bi-functional moleculesynthesis described in said patent application is applicable for thestage 2 synthesis of bi-functional molecules. This variation involvesthe generation of a library of bi-functional molecules, where theindividual bi-functional molecule synthesis comprises the steps of i)providing at least one identifier template comprising a sequence of ncoding elements, complementary to n carrier identifiers, ii) providing aplurality of carriers, wherein each carrier comprises a) at least onecarrier identifier oligonucleotide capable of recognising apredetermined coding element, b) at least one molecule fragment with atleast one reactive group, and c) at least one linker separating the atleast one molecule fragment from the at least one carrier identifier,iii) contacting each of said coding elements with a carrier identifiercapable of recognising said coding element, and iv) obtaining abi-functional molecule comprising covalently or non-covalently linkedmolecule fragments by linking, by means of a reaction involving reactivegroups, two or more molecule fragments, wherein the bi-functionalmolecule is linked by means of a linker to the identifier template orthe complementary template that templated the synthesis of thebi-functional molecule.

The principle may be applied to the generation of encoded molecules aslisted in the present invention, and may involve reactive groupsmentioned in the present invention, including those listed in FIGS. 6,7, and 8.

E. This is a variation of the principle described in (Pedersen et al.(2003) WO03/078625 A2, “An improved method for synthesizing templatedmolecules”). Any embodiment of bi-functional molecule synthesisdescribed in said patent application is applicable for the stage 2synthesis of bi-functional molecules. The variation provides a methodfor synthesising a bi-functional molecule, said method comprising thesteps of: a) providing at least one identifier template comprising oneor more codons (i.e., oligonucleotide sequences complementary to carrieridentifiers), b) providing a first carrier molecule comprising a zippingdomain, said zipping domain comprises a first part of a molecule pair,being capable of reversible interaction with a second part of themolecule pair, c) providing one or more second carriers, comprising azipping domain comprising the second part of said molecule pair andcapable of reacting with the first carrier, d) contacting the componentsof step a), b), and c) with each other under conditions allowingspecific hybridisation of the carriers to the template(s) anddimerization of the two parts of the molecule pair, e) allowing thereactive groups of the first carrier to react with the reactive groupsof the second carrier(s), f) optionally, cleaving one or more linkers,provided that at least one linker remains to connect the moleculefragments (i.e., the encoded molecule) with the template or thecomplementary template, g) obtaining a bi-functional molecule where theidentifier template directed the synthesis of the encoded molecule.

The principle may be applied to the generation of encoded molecules aslisted in the present invention, and may involve reactive groupsmentioned in the present invention, including those listed in FIGS. 6,7, and 8.

F. This is another variation of the principle described in (Liu et al.WO 2004/016767 A2, Evolving New Moelcular Function). Here, the templatedsynthesis is performed with a template that has an “omega” or “O” typearchitecture. This type of template permits distance-dependent nucleicacid-templated reactions to be encoded by bases far removed from theassociated reactive unit. The method involves providing (i) a templatecomprising a first reactive unit associated with a first oligonucleotidecomprising a codon and (ii) a transfer unit comprising a second reactiveunit associated with a second oligonucleotide comprising an anti-codonthat is capable of annealing to the codon. The codon and/or theanti-codon include first and second regions spaced apart from oneanother. The oligonucleotides then are annealed together to bring thereactive units into reactive proximity. When the oligonucleotides annealto one another, the codon (or anti-codon) with the spaced-apart regionsproduce a loop of oligonucleotides not annealed to the correspondinganti-codon (or codon). A covalent bond-forming reaction then is inducedbetween the reactive units to produce the reaction product, the “encodedmolecule”.

G. These approaches are alternative methods for the templated stage 2synthesis, and are described in the FIGS. 2-8, 11 and 13, and aredescribed in detail elsewhere in this application.

H. This is a variation of the approach for stage 2 synthesis, in whichthe template and the carrier molecules do not have to be associated(hybridized) during the reaction of the molecule fragments (Franch etal., WO 2004/083427, “Ligational encoding of small molecules”). In thecourse of the encoding process, a single-stranded product is generated,including both the template and some or all of the carrier molecules.Thus, the molecule fragments, and the reactive groups that react duringthe stage 2 synthesis step, are covalently associated with the template,which allows e.g. a higher temperature to be employed during thereactors step. This may be an advantage for reactions that areparticularly enthalpy-driven.

Bi-Functional Molecule Formation (i.e. Formation of Carriers for Use ina Templated Synthesis Or Formation of the Final Encoded Molecule):

The above methods for carrier synthesis and templated synthesis can becombined in any way. As an example, 3A (subprocess 3: formation ofbi-functional carrier molecules, as described by any of the embodimentsof (Freskgård et al., WO 2004/039825 A2), for example through enzymaticligation of identifiers; followed by direct transfer acylation stage 2synthesis as described in (Bruick et al., Chemistry and Biology, January1996, 3: 49-56), can be applied to the generation of bi-functionalmolecules.

In a preferred embodiment, a one-to-one relationship exists between theidentifiers and molecule fragments. Thus, one specific identifieridentifies (encodes) one specific molecule fragment. However, it is notessential for the invention that such one-to-one relationship exists.For example, during stage 1 synthesis different identifiers may be addedto the same well, wherefore more than one identifier will encode onemolecule fragment. It is still possible, however, to identify themolecule fragment directly from the sequence of the identifier.Likewise, it is possible to add several molecule fragments to the samewell during stage 1 synthesis. In this case, however, the identity ofthe molecule fragment cannot be deduced directly from the sequence ofthe attached identifier, but must be deduced by other means, for examplemass spectrometry. Finally, it is also possible to add the sameidentifier to several wells, or the same molecule fragment to severalwells. In the former case, however, it is not possible to identify themolecule directly from the sequence of the identifier.

Screening Methods Employing Bi-Functional Molecules.

Once the bi-functional molecules have been generated, the desiredmolecules may be identified in any way possible. Thus, a number ofscreening methods exist, for the identification of organic moleculeswith desired characteristics. Different types of selection or screeningprotocols are described in (Liu et al. (2002), WO 02/074929 A2; Pedersenet al. (2002) WO 02/103008 A2; Pedersen et al. (2003) WO03/078625 A2;Lerner et al., EP 0643778 B1, Encoded combinatorial chemical libraries;Dower et al., EP 0604552 B1; Freskgård et al., WO 2004/039825 A2; Morganet al., 2005, WO 2005/058479; Harbury and Halpin, WO 00/23458).

Specific screening methods employing bifuntional molecules for theidentification of organic molecules with desired characteristics includebut are not limited to:

Affinity selection on immobilised target molecules. In this approach thetarget molecules (e.g., DNA, RNA, protein, peptide, carbohydrate,organic or inorganic molecule, supramolecular structure or any othermolecule, is immobilized covalently or non-covalently to a solid supportsuch as beads, the bottom of a well of a microtiter plate, a reagenttube, a chromatographic column, or any other type of solid support. Alibrary of bi-functional molecules are now incubated with theimmobilized target molecule, excess non-bound bi-functional moleculesare washed off by the replacing supernatant or column buffer with buffernot containing bi-functional molecules one or more times. After washingthe bound bi-functional molecules are released from solid support byaddition of reagents, specific ligands or the like that results in theelution of the bi-functional molecule, or the pH is increased ordecreased to release the bound bi-functional molecules, or theidentifier of the bi-functional molecule is cleaved off from the encodedmolecule with a reagent, pH change or light-induced cleavage. Therecovered identifiers can now optionally be amplified by PCR, and clonedand sequenced to reveal the structure of the ligands encoded by theidentifier, or alternatively, be amplified and taken through anadditional round of templated synthesis. As an alternative, theidentifiers or bi-functional molecules comprising identifiers, are notreleased from slid support, but rather the identifiers are optionallyamplified by PCR directly while still immobilised on solid support.

Affinity selection on target molecules in solution, followed by anymeans of isolation of the bi-functional molecules bound to the target,e.g. by immunoprecipitation of the target-bi-functional moleculecomplexes. A library of bi-functional molecules are incubated withtarget molecules (e.g. a protein). After complex formation ofbi-functional molecules with target, the complex is isolated fromnon-complexes, for example by the addition of polyvalent antibodiesagainst the target molecule and precipitation ofantibody-target-bi-functional molecule complexes, or is precipitated bythe addition of beads that bind the target molecules. The latter may forexample be by addition of streptavidin-coated beads that bind topre-biotinylated targets. The identifiers recovered by precipitation cannow be characterised or amplified, e.g., by PCR, as described in (i).The sequence of the identifiers will reveal the identity of the encodedmolecules that bind the target molecules.

Affinity selection on target molecules in solution, followed by gelretardation, chromatographic separation e.g. size exclusionchromatography, or separation by centrifugation e.g. in aCsCl₂-gradient. A library of bi-functional molecules are incubated withtarget molecules (e.g. a protein). After complex formation ofbi-functional molecules with target, the complex is isolated fromnon-complexes, for example by gel electrophoresis or size exclusionchromatography, or any other chromatographic or non-chromatographicmethod that separates the target-bi-functional molecule complexes fromnon-complexed bi-functional molecules, for example based on thedifference in size and/or charge. The identifiers of the bi-functionalmolecules of the column fraction or band on the gel that comprisestarget-bi-functional molecule complexes are now characterised oramplified, e.g., by PCR, as described above. The sequence of theidentifiers will reveal the identity of the encoded molecules that bindthe target molecules.

Affinity selection on surfaces. Particles, preferably small particles,of solid material, e.g., metal particles, metal oxide particles, grindedplastic, wood, preformed carbon nanotubes, clay, glas, silica, bacterialbiofilm or biofilm of other microorganism, cement, solid paintparticles, laminate, stone, marble, quartz, textile, paper, skin, hair,cell membranes, industrial membranes, epiderm, or the like, is added toa solution comprising a library of bi-functional molecules. Afterincubation, one or more washing steps are performed, to remove unboundbi-functional molecules. Then, the bi-functional molecules bound to thesurface, or the identifiers of the bi-functional molecules bound to thesurface, are released as described above, and the identifierscharacterised and/or amplified as described above. Selection forintracellularisation. Bi-functional molecules are incubated with cellsor micelles, or on one side of a lipid membrane, or on one side of acell monolayer (e.g. CaCo2 cell monolayer), in order to allow thebi-functional molecule to pass or become immobilized into the membranes.Then, a number of washing steps are performed in order to removebi-functional molecules that have not become immobilized or have passedthe membrane. Identifiers from bi-functional molecules that have becomeimmobilized or have passed the membrane are now amplified and/orcharacterised as described above. The encoded molecule of bi-functionalmolecules that have either become immobilized in the membrane or havepassed the membrane, represent potential transporters forintracellularization, i.e. by attaching these encoded molecules (withoutthe oligonucleotide tag) to e.g. non-oral drugs these may become orallyavailable, because the transporter mediate their transport across thecell.

Selection by phase partitioning. A two- or three phase system may be setup, wherein the bi-functional molecules will partition out according (atleast in part) to the characteristics of the encoded molecules.Therefore, the principle allows the identification of encoded moleculesthat have particular preference for a certain kind of solvent. Again,the identifiers of the isolated bi-functional molecules can be amplifiedand/or characterised after the selection has occurred. It may benecessary to coat the nucleic acid component of the bi-functionalmolecule with e.g. DNA binding proteins, in order to ensure that thepartitioning of the bi-functional molecule is significantly correlatedwith the characteristics of the encoded molecule of the bi-functionalmolecule.

Selection for induced dimerisation of target molecules. In a preferredembodiment, encoded molecules are sought that induce the dimerization oftarget molecules. For example, small molecules with the potential toinduce dimerization of protein receptors in the cell membrane may beapplicable as therapeutics. Thus, a selection protocol for encodedmolecules with the potential to induce dimerization of proteins A and Bis a s follows: A library of bi-functional molecules are incubated withproteins A and B. After incubation, the solution is applied to gelelectrophoresis, ultracentrifugation (e.g. CsCl-centrifugation), sizeexclusion chromatography, or any other kind of separation that separatesthe protein A-protein B-bi-functional molecule-complex from un-complexedprotein A and B, and other undesired complexes, such as proteinA-protein B-complex. Bi-functional molecules from the band or fractioncorresponding to the size and/or charge of the protein A-proteinB-bi-functional molecule-complex is recovered, and template identifiersare then amplified and/or characterised as described above. In thiscase, the encoded molecule would be resynthesized, and tested in aprotein dimerisation assay for its effect on the dimerisation of proteinA and B.

Selection by iterative rounds of binding and elution. This is amodification of the methods reported previously (Doyon et al. (2003), 3.Am. Chem. Soc., 125, 12372-12373). Bi-functional molecules are incubatedwith e.g. immobilised target molecule, e.g. a biotinylated enzymeimmobilised on streptavidin beads. After washing one or more times, thebound bi-functional molecules are released from solid support by achange in pH, or by addition of an excess of ligand that binds thetarget molecule (the ligand can be e.g. a small molecule, peptide, DNAaptamer or protein that is known to bind the target molecule).Alternatively, the bi-functional molecules may be released bydegradation of the immobilised target (e.g. by nuclease or protease),denaturation of target or induced conformational changes in targetstructure or the like. The recovered bi-functional molecules are nowre-applied to e.g. immobilised target molecule, optionally after removalor degradation of the ligand or reagent used for elution in the previousstep. Again, washing is performed, and the bound bi-functional moleculeseluted. The process of incubation and binding, washing and elution canbe repeated many times, until eventually only bi-functional molecules ofhigh affinity remains. Then the identifiers of the bi-functionalmolecules are amplified and/or characterised. Using this kind ofiterative binding and elution, enrichment factors higher than100.000-fold can be obtained.

Targets may be immobilised on columns, on beads (batch selection), onthe surface of a well, or target and ligands may interact in solution,followed by immunoprecipitation of the target (leading toimmunoprecipitation of ligands bound to target).

Screening in compartments. A library of template identifiers areincubated with lipids, bi-functional carrier molecules, primers,nucleotides and other components necessary for micelle formation and PCRamplification, transcription, stage 2 templated synthesis, and assay.The lipids are allowed to assemble into micelles; the number of inputtemplates are adjusted so that on average every fifth micelle contains atemplate after micelle formation. A PCR reaction is performed, togenerate multiple copies of the same template in each micelle. Thentranscription is performed, in order to generate many copies of thecorresponding single-stranded RNA. The carriers now hybridise to theRNA, and reaction between reactive groups of the carriers take place, toform the same encoded molecule in multiple copies in a given micelle.Finally, the assay is performed (for example, an enzyme assay thatexamines the ability of the encoded molecule to inhibit enzymeactivity). Positive micelles are picked by hand under microscope, orsorted using e.g. a FACS sorting machine. Then the identifiers containedwithin the positive micelles are amplified and/or characterised asdescribed above. Encoded molecules identified in this way representpotential inhibitors of the enzyme; after resynthesis of the freeencoded molecule, the encoded molecule can be examined for itsinhibitory effect on enzyme activity in standard inhibition assays.

Whole organism selection. A library of bi-functional molecules,optionally modified by e.g. coating proteins, is injected into a dead orliving animal, for example a mouse. After incubation for a period oftime (e.g. two hours) in the animal, specific tissue or organs arerecovered, and the bi-functional molecules associated with specificorgans can be characterised, by e.g. PCR amplification and/or sequencingof the corresponding identifiers. As a specific example, a mousecarrying a tumor can be injected with a library of bi-functionalmolecules. After incubation, the tumor can be isolated from the animal.The bi-functional molecules associated with the tumor are potentialtherapeutics or diagnostics for that cancer.

Any other kind of selection or screening which may be performedemploying libraries of bi-functional molecules.

The abovementioned target molecules may be any supramolecular structure(e.g. nanoclusters, multiprotein complex, ribosomes), macromolecule(e.g. DNA, RNA, protein, polymers such as carbohydrates, thiophenes,fibrin), or low molecular weight compound (e.g. cAMP, small peptidehormones, chelates, morphine, drug).

After having performed any of the selections above, the identifiers ofthe output bi-functional molecules can be amplified, and taken throughone more round of stage 2 synthesis. Then, the same or another selectionprotocol can be performed. This process can be repeated until anappropriately small number of different bi-functional molecules arerecovered.

Any combination of stage 1 and stage 2 synthesis may be employed in thegeneration of bi-functional molecules. Moreover, any screening methodmay be combined with any combination of stage 1 and stage 2 synthesisschemes. Thus, referring to the numbering above for stage 1 synthesisprocedures (1, 2, 3, . . . X) and stage 2 synthesis procedures (A, B, .. . Z) and screening methods (i, ii, iii, . . . n), the followingcombinations may be employed during the generation and use ofbi-functional molecules:

1Ai, 1Aii, . . . , 1An, 1Bi, 1Bii, . . . 1Bn, . . . 2Ai, . . . , XZn

Furthermore, any sequence of encoding and screening schemes may beapplied:

1Ai+1Ai, 1Ai+1Aii, . . . , XAn+XAn, 1Bi+1Bi, . . . XZn+XZn (all of whichrepresent two rounds of library generation and screening), and1Ai+1Ai+1Ai, 1Ai+1Ai+1Aii, . . . , XZn+XZn+XZn (all of which representthree rounds of library generation and screening), andany sequence of, and any number of, library generation and screeningrounds.

Finally, in each of the stage 1, stage 2 and screening steps, one ormore approaches may be employed. For example, carriers may be generatedduring stage 1 synthesis, by any combination of approaches 1−X. Thecarriers may likewise be employed in any combination of stage 2synthesis approaches. Finally, the bi-functional molecules generated maybe screened by any combination of screening approaches i-n. Again, anysequence of encoding and screening schemes may be applied.

In a preferred embodiment, the stage 1 carrier synthesis is onlyperformed once, whereas the stage 2 synthesis is performed any number oftimes. In this case, each of the stage 2 syntheses may employ thecarriers synthesised in the beginning. Thus, a preferred sequence ofstage 1 library synthesis, stage 2 library synthesis and libraryscreening is as follows:

5Ai+Ai, where firstcarrier synthesis is performed according to (5) above, where theidentifiers are ligated together chemically, followed bytemplated synthesis as described in (A) above, and using the carriersgenerated immediately above, followed byscreening of the library generated immediately above, by affinityselection on the target molecule as described in (i) above, followed byPCR amplification of the DNA part of the bi-functional moleculesrecovered from this affinity selection, followed bytemplated synthesis as described in (A) above, using the PCR productgenerated immediately above as DNA templates, and using the carrierssynthesised in the first part of the process, and finally followed byscreening of the library generated immediately above, by affinityselection on the target molecule as described in (i) above,to identify bi-functional molecules with affinity for the targetmolecule.

Finally, the encoded molecules with the desired characteristics(affinity for the target molecule) may be identified, by PCRamplification and sequencing of the DNA templates of the recoveredbi-functional molecules.

In another preferred embodiment, three rounds of library synthesis isperformed, where the first library is generated solely by stage 1synthesis, the second library is made through a combination of carriersynthesis and templated synthesis, and the third library is made throughtemplated synthesis using carriers synthesised in the previous round oflibrary synthesis. One such sequence of library syntheses and screeningis 5iv+5Aiv+Aiv: First a library of bi-functional molecules is madewhere four molecule fragments are linked together to form the encodedmolecule, and four identifiers are chemically ligated together to formthe DNA template, as described in (5) above, followed by affinityselection on a surface (for example a suspension of metal-oxideparticles in aqueous buffer) as described in (iv) above, followed by

Carrier synthesis as described in (5) above, where two moleculefragments are linked together and two identifiers are ligated together,to form carrier molecules consisting of two molecule fragments and twoidentifiers, followed byTemplated synthesis, where first the DNA templates of the recoveredbi-functional molecules from the screening (iv) above are PCR-amplified,and the templates used in a templated synthesis using the carriersgenerated immediately above. The choice of identifiers and moleculefragments must ensure that an encoded molecule recovered from the firstscreening round is amplified and turned into a number of copies of thesame encoded molecule, attached to a DNA template, even though differentmethods are used in the first and second round of library generation(i.e., 5 and 5A, respectively). Thus, the final bi-functional moleculesgenerated either initially or after the amplification of recoveredtemplates, contain identical encoded molecules, but may carry differentDNA templates. Library generation is followed byAffinity selection on a surface (in this example, a suspension ofmetal-oxide particles in aqueous buffer) as described in (iv) above,followed byTemplated synthesis, where first the DNA templates of the recoveredbi-functional molecules from the screening (iv) immediately above arePCR-amplified, and the templates used in a templated synthesis using thecarriers generated above, followed byAffinity selection on a surface (in this example, a suspension ofmetal-oxide particles in aqueous buffer) as described in (iv) above.

Finally, the encoded molecules with the desired characteristic (affinityfor the surface) may be identified, by PCR amplification and sequencingof the DNA templates of the recovered bi-functional molecules.

Reactive groups may be protected and de-protected at various stepsduring the synthesis of the encoded molecules, in order to ensure thatthe reactions proceed in the desired order, and links desired moleculefragments in the desired way. Also, before, during or after linkage ofthe molecule fragments, individual parts of the encoded molecule may bemade cyclic, by reaction of reactive groups of different moleculefragments, or by reaction of reactive groups within a molecule fragment.

After or during stage 1 or stage 2 synthesis, the encoded molecule maybe modified, for example by hydrogenation, reduction/oxidation, or byreaction with any chemical moiety. If the modification is done on thepool of bi-functional molecules, the modification is typically notencoded (i.e., no identifiers are added). As for the stage 1 and stage 2syntheses, it is important that the conditions and reagents of thechemical reaction on the encoded molecule does not modify the templateor complementary template to such an extent that the template orcomplementary template cannot be amplified and/or sequenced. Examples ofreactions and reagents that may be applied for the modification of theencoded molecule are shown in FIGS. 6 and 7.

The split and mix process (Stage 1) that leads to the formation ofbi-functional carrier molecules may involve any number of rounds.Preferably the number of rounds is between 1 and 10. Likewise, theDNA-templated synthesis (Stage 2) may involve templates, each of whichthat hybridise to any number of carrier molecules. Preferably, atemplate can hybridise to 1-10 carrier molecules simultaneously. Anytemplate, however, has a given maximum of carriers that it can bind. Thecarriers may be annealed to the template and reacted two at a time,three at a time, four at a time, more than four at a time, or all atonce, or one carrier may be reacted with a reactive group on thetemplate (i.e. the carriers are annealed and reacted one at a time, whenthe nascent encoded molecule is linked to the template). Any combinationof hybridisations and reactions may be used until the reactive groupshave reacted and the encoded molecule formed.

During stage 1 synthesis, a repertoire of molecule fragments can containdifferent kinds of reactive groups that participate in differentcoupling reactions. As an example, the first molecule fragment reactedwith the functionality on the linker L may be an amino acid.

After coupling to the linker, an amine NH₂ functionality may beavailable for reaction with the incoming molecule fragment. Thus, theincoming molecule fragment must contain a reactive group capable ofreacting with the amine. Example reactive groups of the incomingmolecule fragment are carboxylic acid COOH (reacts through an acylationreaction with the amine by addition of e.g. EDC/NHS), aldehyde CHO(reductive amination reaction with amine), sulfonate (alkylationreaction with amine), sulfonoyi chloride (sulphonamide formation withamine), and substituted aromates (nucleophilic aromatic substitution).During stage 1 synthesis each molecule fragment is added to a specificwell, and therefore, the reaction conditions that are ideal for aparticular reaction may be employed in that specific well.

Likewise, during stage 2 synthesis, carriers carrying a variety ofdifferent molecule fragments can be employed. For example, if a libraryof templates all bind one carrier containing an amine reactive group,the other carrier can contain for example a COOH, CHO, sulfonoylchloride or a substituted aromate. The reactions of stage 2 synthesismay not be separated in separate wells, and therefore, the moleculefragments must be compatible with the reaction conditions andreagents/catalysts used for all reactions performed. The reactions canbe performed simultaneously if the reactions can be performed under thesame conditions, or the reactions may be performed sequentially if thereactions require different conditions and reagents/catalysts.

For both stage 1 and stage 2 synthesis, the number of reactive groups Xon the linker, or the number of reactive groups on the nascentbi-functional molecule can vary. If the linker has one reactive group,the molecule fragment that becomes attached to the linker must have atleast one reactive group in order to attach further molecule fragmentsto the nascent bi-functional molecules. If all molecule fragmentscontain two reactive groups, one reactive group can react with thenascent bi-functional molecule, and one may react with the next moleculefragment that is attached. The resulting structure is a linearstructure, like beads on a string. Alternatively, if the nascentbi-functional molecule carries three or more reactive groups, onereactive group may be used for attachment to the nascent bi-functionalmolecule, and the remaining reactive groups may be attached to differentmolecule fragments (in the same or in different synthesis rounds), andthe resulting structure is branched. Molecule fragments containing morethan two reactive groups are termed scaffolds; the structure generated(where at least two encoded molecule fragments in addition to thenascent bi-functional molecule become attached directly to the samemolecule fragment) is termed a scaffolded or branched molecule. Typicalscaffolds include aromatic structures, benzodiazepines, hydantoins,piperazines, indoles, furans, thiazoles, steroids, diketopiperazines,morpholines, tropanes, coumarines, qinolines, pyrroles, oxazoles, aminoacid precursors, cyclic or aromatic ring structures, and many others,all of which must contain at least three reactive groups in order to beconsidered scaffolds.

If two or more reactive groups of a molecule fragment react directlywith the nascent bi-functional molecule, a cyclic structure results.

The molecule fragment repertoires of different rounds of stage 1synthesis may be of different size, i.e. a different number of moleculefragments may be employed in different rounds. The identifiers added indifferent wells or different rounds of stage 1 synthesis may be ofdifferent length and/or composition. In a preferred embodiment one wellcontains one specific identifier and one specific molecule fragment.However, in another preferred embodiment more than one molecule fragmentis added to each well. This will result in relaxation of theone-template/one-encoded molecule relationship that exist betweentemplate sequence and encoded molecule, and can therefore only bepursued to a limited extent. Likewise, in another preferred embodimentmore than one identifier sequence is added to each well. If the sameidentifier is added to different wells containing the same moleculefragment, this will also result in relaxation of theone-template/one-encoded molecule relationship.

Under certain circumstances, the templated synthesis (Stage 2) may berepeated using the bi-functional molecules generated in the templatingprocess as carriers in a second or third templating process. As anexample, a templated synthesis reaction is employed that involvesligation of the two carrier molecules, to form a bi-functional moleculein which all the sequence-units identifying the molecule fragments arecovalently linked, and where the encoded molecule remains attached tothe DNA portion (for an example, see FIG. 4, example 2). Then thebi-functional molecule resulting from the templated reaction (in FIG. 4,example 2, the DNA template attached to the encoded molecule XY), may beused in a next templated reaction, using a template that carries twosequence units, one of which is complementary to the DNA portion of thebi-functional molecule generated in the first templated reaction.

Under certain circumstances, it may be desirable to i) generate thebi-functional molecules used in the screening process through split andmix synthesis only, e.g. exclude the stage 2 templating process in theinitial bi-functional molecule generation. If desired, recoveredbi-functional molecules from the screen may then be amplified using acombination of split and mix synthesis (stage 1) and templated synthesis(stage 2) (see for example FIG. 2), using the template generated duringthe bi-functional molecule synthesis described in i) above.

In i) above, a template is generated using a split and mix procedure,involving also the synthesis of an encoded molecule. However, a simplerversion of this split and mix approach can be applied to the generationof a library of DNA templates. As an example, to make a library of n³DNA templates where each DNA template carries 3 codons, and where eachof the 3 positions can contain any of n different codons, perform thefollowing steps: i) Add an aliquot of a mixture of n position1 codon DNAduplexes to each of n wells, ii) To each well, add a specific position2codon duplex DNA, iii) Ligate the position 1 codon DNA duplex and theposition2 codon DNA duplex (f.ex. by the use of a DNA ligase, or anactivated phosphate (e.g. Imidazole-phosphatexxxxx, see ref . . . ), iv)Mix the contents of all wells, v) Add an aliquot of the mixture ofligated position1-position2 DNA duplexes to each of n wells, vi) To eachof the n wells, add a specific position3 codon DNA duplex, vii) Ligatethe position3 duplex to the positionlposition2 duplexes, viii) Mix thecontents of all wells.

The resulting pool contains n³ different DNA templates, carrying ndifferent codons at each of 3 codon positions. The DNA duplexes carryingposition1 or position3 codons may also carry primer binding sites, toallow amplification by PCR of the generated templates.

The nucleic acid carrying the codons may be RNA, DNA or any other typeof nucleic acid or nucleic acid derivative, and it could be single- ordouble-stranded.

It may be desirable, after the synthesis of the bi-functional moleculesbut before the bi-functional molecules are used in a selection orscreening experiment, to make the template double-stranded. This may bedone by annealing a primer to the template strand (or its complementarystrand), and extend for example using sequenase. This will form a doublestranded oligonucleotide template, which is expected to be more inert inthe selection experiment than the corresponding single-strandedoligonucleotide, because of the single-stranded oligonucleotide'sability to bind the target as an aptamer.

In a preferred embodiment, the carrier molecules are immobilised duringtheir synthesis. For example, the following types of solid support maybe employed for the immobilisation: Sepharose beads with functionalitieslike-SH, —COOH or —NH₂ groups (where the identifiers or templates arecovalently coupled by e.g. disulfide formation, acylation, andacylation, respectively); tentagel beads with functional groups e.g.—SH, —COOH or —NH₂ groups; streptavidin coated beads (where the carriersor templates are covalently coupled to biotin, which in turn can benon-covalently bound to streptavidin); and many other types of supportsand functionalities (e.g. polystyrene, polypropylene, agarose (e.g.Hispanagar); diol functionalities, ester functionalities; amidefunctionalities, glyoxal functionalities). Advantages of immobilisationof carriers or templates include the easy isolation of single-strandedcarriers or single-stranded templates (for use in stage 2 synthesis),easy removal of excess reagents (molecule fragments, identifiers,catalysts, reactants), the ability to conveniently change solvent (forexample, change to organic solvent prior to the reaction of the moleculefragments), removal of protection groups, etc.

Finally, the molecule fragments may be immobilised prior to reactionwith the growing carrier molecule. This allows for the purification ofgrowing carriers that have reacted with the immobilised moleculefragments. This in turn allows purification of full-length carriers(i.e., carriers that contain the desired molecule fragments), which willlead to a more efficient stage 2 synthesis.

The carriers and/or templates may be released from the solid support byhydrolysis (e.g. high pH), proteolysis (of a peptide linker), thiolmediated cleavage of disulfide bond, nuclease-mediated cleavage, etc.

Types of Encoded Molecules:

Different kinds of molecules may be generated and attached to the DNAtemplate that encodes it. Molecules that may be generated by the presentinvention include small compact molecules, linear structures, polymers,polypeptides, poly-ureas, polycarbamates, scaffold structures, cyclicstructures, natural compound derivatives, alpha-, beta-, gamma-, andomega-peptides, mono-, di- and tri-substituted peptides, L- and D-formpeptides, cyclohexane- and cyclopentane-backbone modified beta-peptides,vinylogous polypeptides, glycopolypeptides, polyamides, vinylogoussulfonamide peptide, Polysulfonamide conjugated peptide (i.e., havingprosthetic groups), Polyesters, Polysaccharides, polycarbamates,polycarbonates, polyureas, poly-peptidylphosphonates, Azatides, peptoids(oligo N-substituted glycines), Polyethers, ethoxyformacetal oligomers,poly-thioethers, polyethylene, glycols (PEG), polyethylenes,polydisulfides, polyarylene sulfides, Polynucleotides, PNAs, LNAs,Morpholinos, oligo pyrrolinone, polyoximes, Polyimines,Polyethyleneimine, Polyacetates, Polystyrenes, Polyacetylene, Polyvinyl,Lipids, Phospholipids, Glycolipids, polycycles, (aliphatic), polycycles(aromatic), polyheterocycles, Proteoglycan, Polysiloxanes,Polyisocyanides, Polyisocyanates, polymethacrylates, Monofunctional,Difunctional, Trifunctional and Oligofunctional open-chain hydrocarbons.

Monofunctional, Difunctional, Trifunctional and OligofunctionalNonaromat Carbocycles, Monocyclic, Bicyclic, Tricyclic and PolycyclicHydrocarbons, Bridged Polycyclic Hydrocarbones, Monofunctional,Difunctional, Trifunctional and Oligofunctional Nonaromatic,Heterocycles, Monocyclit, Bicyclic, Tricyclic and PolycyclicHeterocycles, bridged Polycyclic Heterocycles, Monofunctional,Difunctional, Trifunctional and Oligofunctional Aromatic Carbocycles.Monocyclic, Bicyclic, Tricyclic and Polycyclic Aromatic Carbocycles

Monofunctional, Difunctional, Trifunctional and Oligofunctional AromaticHetero-cycles. Monocyclic, Bicyclic, Tricyclic and PolycyclicHeterocycles. Chelates, fullerenes, and any combination of the above andmany others. A non-comprehensive list of example generic and specificstructures generated by the present invention is shown in (FIG. 13).Each of the molecule fragments linked together during the process may beprepared by any kind of synthetic protocol, including standard organicsynthesis, prior to the library generation process. Therefore, any typeof chemical moiety can be included in a molecule or library of moleculesof the present invention. The linkages (bonds) between the moleculefragments (the linkages that are generated during the library generationprocess) must be compatible with the presence of the DNA.

The types of bonds that may be generated must be compatible with thepresence of the oligonucleotide. Types of linkages that may be generatedin the present invention include amide bonds, carbamates, sulfones,sulfoxides, phophodiester bonds, carbohydrate bonds, ureas,phosphonates, esters, and many others A non-comprehensive list of suchlinkage bonds is shown in (FIGS. 6 and 7), and is listed within thedescription below.

Following the encoded synthesis of the molecules (whereby one or morebi-functional molecules have been formed), the encoded molecules may bemodified in a non-encoded way. For example, the library of molecules maybe hydrogenated, acylated, oxidised or reduced, or protection groups maybe removed. It will often be necessary to protect chemical motifs thatwould otherwise participate in the reactions that attach moleculefragments, either through reaction with molecule fragments, reagents, orother nascent bi-functional molecules. These chemical motifs can then begenerated at the end of library synthesis, by deprotection reactionsafter the encoding- and encoded reactions. In principle, any of thereactions listed in FIG. 6, 7 or 8 may be used after the encodedsynthesis, in order to modify the encoded molecules.

Use of the method for the synthesis of specific (one or a few) differentmolecule species. The described methods for organic molecule synthesismay be applied to a) synthesis of compounds of high stereochemicalpurity, or b) synthesis of compounds without the cumbersome use ofprotection groups, or c) as a means to increase the yield of synthesissteps that are usually inefficient, for example because the necessaryconcentrations cannot be achieved in a standard organic synthesissetting.

Examples of such uses of the methods is synthesis of a sequence ofsaccharides, without the use of protection groups.

Use of the Library for Screening.

The bi-functional molecules generated by the present invention may beused to identify encoded molecules with particular characteristics. Forexample, the bi-functional molecules may be employed in affinityselection experiments, in which bi-functional molecules capable ofbinding to proteins, DNA, RNA, surfaces, inorganic or organic molecules,and other molecules and substances may be identified. During theaffinity selection experiments bi-functional molecules interacting withthese molecules or substances may be isolated, and identified bysequencing the DNA portion of the bi-functional molecules.Alternatively, the bi-functional molecules may be screened for catalyticactivity, for the ability to interact with other bi-functionalmolecules, for the ability to become internalised into a cell, for theability to interfere with conductance or fluorescence or any othercharacteristics of another molecule or substance, or for any othercharacteristics desired. Finally, the library may be screened for theability to interact with library members of other types of molecules,for example phage-displayed peptides or proteins, in order to identifybi-functional molecules that interact with peptides or proteins of thephage-display library.

The DNA portion of the bi-functional molecules in these different typesof screens and selections allows the easy and highly sensitiveidentification of the encoded small molecule component responsible forits isolation during the screen or selection. In principle, one speciesof a bi-functional molecule is enough to allow its identification, byfirst amplifying the DNA component by PCR, followed by sequencing of theDNA.

In addition, the DNA component allows several rounds of screening andamplification to be performed. Thus, after a screening round, therecovered population of bi-functional molecules may be amplified (byamplification of the DNA template, followed by Stage 1 carrier synthesis(or the original preparation of carriers may be used) and Stage 2templated synthesis using the amplified DNA template for bi-functionalmolecule synthesis). The amplification generates several copies of eachof the recovered bi-functional molecules without the need foridentification of the recovered bi-functional molecules. The ability toperform several rounds of screening or selection allows efficientscreening of very large libraries of bi-functional (OBS) molecules,involving up to at least 10¹⁶ different bi-functional (OBS) molecules.

A typical selection protocol involves the addition of a population (alibrary) of bi-functional (OBS) molecules to an affinity column, towhich a certain molecular target (e.g., a receptor protein or a DNAfragment) has been immobilised. After washing the column, the bindersare eluted. This eluate consists of an enriched population ofbi-functional molecules with affinity for the immobilised targetmolecule. The enriched population may be taken through an amplificationround (by first amplifying the template and then using the amplifiedtemplate in a stage 2 templated synthesis of bi-functional molecules),and then be subjected to yet a selection round, where the conditionsoptionally may be more stringent. After a number of suchselection-and-amplification rounds, an enriched population ofhigh-affine binders are obtained. A typical selection process isillustrated in FIG. 9. Other types of selection methods that may be usedinclude immunoprecipitation, FACS sorting, mass spectrometry,cell-surface subtraction, in vivo selection (e.g., injection ofbi-functional (OBS) molecules into animals and isolation ofbi-functional (OBS) molecules from specific tissues) and gel mobilityshift assays.

Polyvalent display and other means of increasing the likelihood ofidentifying encoded molecules with weak characteristics.

Under certain conditions the requirements of an encoded molecule, inorder to be isolated during the screening step, are too strong, and fewor none of the encoded molecules of a library are expected to fulfil therequirements. Such requirements may be for example high affinity or highcatalytic turn-over.

In those cases it may desirable to employ a multivalent display mode,i.e., to generate libraries of multivalent encoded molecules (eithermultiple encoded molecules attached to multiple identifiers, or multipleencoded molecules attached to one identifier). During a selection stepin which for example an encoded molecule interacts weakly with a targetprotein, a multivalent encoded molecule may interact with multipleprotein targets through the multiple copies of encoded molecules that itcontains, and as a result, may bind with higher affinity because of theavidity effect. Likewise, in a screening or selection step for catalyticefficiency, a multivalent encoded molecule may generate more product ina given time, and may be isolated because of this.

A preferred means of generating libraries of multivalent encodedmolecules each containing multiple copies of the same encoded molecule,is as follows (FIG. 11A). First, a library of templates that may be usedin stage 2 synthesis is generated. The library of templates may begenerated as described in FIG. 5, whereafter the libraries are thencircularised (for example by ligating the two ends of a template).Alternatively the circular templates can be generated as described inFIG. 4, example 5. A rolling circle amplification is hereafter performedon the library of templates, leading to the generation of a library oftemplates, where each template now contains multiple copies of thesequence that may be used in stage 2 synthesis to generate an encodedmolecule. Secondly, templated synthesis is performed using the templatesgenerated (using carriers optionally generated by stage 1 synthesis, orgenerated by any other means), leading to multivalent encoded moleculeseach containing multiple copies of an encoded molecule. Multivalentencoded molecules containing multiple copies of two different encodedmolecules may be generated by ligating together sequences two-and-twobefore circularisation and rolling circle amplification (i.e., ligatetwo templates together, circularise the ligation product, and performrolling circle amplification). After templated synthesis on thesetemplates, the library will consist of bi-functional molecules each withmultiple copies of two different encoded molecules.

The multivalent encoded molecules can now be used in various screeningor selection processes. For example, the multivalent encoded moleculesmay be added to an affinity column, to which target protein has beenimmobilised with an appropriately high density, so that multivalentencoded molecules may interact with several immobilised targetssimultaneously. This will lead to the isolation of bi-functionalmolecules that contain encoded molecules with affinity for theimmobilised target protein.

Divalent encoded molecules (bi-functional molecules containing twocopies of an encoded molecule) may be generated in several differentways. In a stage 1 synthesis where the linker molecule contains tworeactive groups, the stage 1 synthesis may lead to the formation of twoencoded molecules (FIG. 11B). Hereafter, the divalent carrier moleculescan be used in a templated stage 2 synthesis scheme, for generating alibrary of divalent template encoded molecules (FIG. 11C). Theseprinciples for the formation of divalent encoded molecules may of coursebe applied to the generation of trivalent and higher valency encodedmolecules, by employing linkers during stage 1 carrier synthesis thatcarry three or more, respectively, reactive groups.

The divalent encoded molecules generated may be used in screening orselection experiments. For example, a library of divalent encodedmolecules may be added to beads to which a target molecule has beencoupled with appropriately high density, and an affinity selectionexperiment performed, leading to the isolation of divalent encodedmolecules which contain encoded molecules with affinity for the targetmolecule. Divalent encoded molecules may be particularly advantageous touse when selecting for affinity to a homodimeric target molecule, or anyother target that contains two or more identical binding sites. Relevanttargets include membrane proteins such as the Epo-receptor, p53, HER2,Insulin Receptor, many interleukins, palindromic DNA- or RNA-sequences,or fibrin. Divalent encoded molecules containing identical encodedmolecules are also appropriate for affinity selection on targetmolecules with one binding site, where the binding site is partly orfully symmetrical, and therefore allows two identical encoded moleculesto interact.

A similar principle may be applied to the generation of bi-functionalmolecules that carry a helper moiety. For example, when searching for anencoded molecule with affinity for a particular nucleic acid sequence,it may be advantageous to generate a bi-functional molecule thatcontains a nucleic acid sequence that is complementary to the sequencenext to the target nucleic acid sequence, and in this way increase thetotal affinity of the bi-functional molecule for the target nucleic acid(FIG. 11D). A similar approach may be applied to the isolation ofencoded molecules with affinity for any target molecule with two bindingsites, or a binding site that can accommodate two binding moieties.Thus, as an example, if a ligand is known for a binding site in aprotein, this ligand may be coupled to the bi-functional molecule, inorder to guide the encoded molecule to the target protein, and in orderto increase the affinity of the bi-functional molecule (carrying theknown ligand) for the target protein (FIG. 11E). A simple way ofattaching the known ligand is by hybridisation, i.e. the encodedmolecule is linked to the template and the known ligand is linked to anoligonucleotide that is complementary to part of the template (FIG. 11F)Similar approaches may be used for isolation of encoded molecules withaffinity for a target binding site, where the binding site can beoccupied by both the encoded molecule and the known ligandsimultaneously (FIG. 11G). Finally, it may be desirable to increase theoverall affinity of the bi-functional molecule for the target by linkinga short oligonucleotide that is complementary to the template of thebi-functional molecule to the target. The short oligonucleotide willthen function as a helper moiety that increases the affinity of thebi-functional molecule for the target, by hybridisation of the shortoligonucleotide to the bi-functional molecule (FIG. 11H).

Selections employing such bi-functional molecules to which have beenattached a helper moiety may be applied to affinity selection againstall kinds of targets, including protein-heterodimers as well asprotein-homodimers, and thus molecular targets include HER2,Insulin-receptor, VEGF, EGF, IL-4, IL-2, TNF-alpha, the TATA-box ofeukaryotic promoter regions, and many others.

Dynamic combinatorial library of dimers or trimers of encoded molecules.

The bi-functional molecules of a library may be designed in a way thatleads to transient complex formation between 2, 3, or more bi-functionalcomplexes during the screening process. This may be desirable,especially in cases where the libraries that have been generated arerelatively small, or in cases where it is desirable to screen a largenumber of combinations of encoded molecules for synergistic effects. Inorder to generate transient complexes, the bi-functional molecules maybe designed so as to comprise half of a transient interaction pair. Forexample, a short single stranded oligonucleotide region may be includedin the design of the identifiers of the bi-functional molecules thatresult from the present invention; if some of the bi-functionalmolecules carry a molecular entity “A” and some other bi-functionalmolecules of the library carry another molecular entity “B” thatinteracts transiently, i.e. forms a short-lived complex with, “A”, thenthe two sets of bi-functional molecules of the library will formtransient dimers of bi-functional molecules. These transient dimers maythen be exposed to a screening process, for example affinity selection,where the dimers are then examined for ability to bind to a certaintarget. As an example, for each of the species of bi-functionalmolecules, half of the generated bi-functional molecules carry the oligosequence 3′-ATGC-5′ in the proximity of the encoded molecule, and theother half of the generated bi-functional molecules carry the oligosequence 3′-GCTA-5′. When all the generated bi-functional molecules areincubated at appropriately low temperature, different combinations ofdimers will transiently form, and allow for a feature displayed by thecombination of the corresponding two encoded molecules to be selectedfor. This feature could be the binding of the two encoded molecules ofthe dimer to bind simultaneously to a target molecule. If appropriatelydesigned, trimers may be (transiently) formed, by formation of triplexDNA between three bi-functional molecules. In this way, all the possibledimers (or trimers) of a pool of bi-functional molecules may be screenedfor the desired feature. See (FIG. 14).

Molecular biological methods applicable to bi-functional molecules.

As the present invention involves the templated synthesis of encodedmolecules, most in vitro molecular biological techniques may be appliedto the DNA-, RNA- or any other oligonucleotide-portion of the template,and as a result of the encoding by this template, indirectly may beapplied to the encoded molecule. Examples of such molecular biologicaltechniques applicable to the encoded molecules of this invention arefisted in FIG. 10.

Characterisation of encoded molecules identified during a screening of alibrary of bi-functional molecules.

Once the screening of a library of bi-functional molecules have beendone, the isolated bi-functional molecules may be identified by cloningof the oligonucleotide portion, and sequencing. The sequencing may bedone by any means, including Sanger sequencing, mass spectrometry-basedsequencing, single molecule sequencing, or sequencing by hybridisationto oligonucleotide arrays. The characteristics of the encoded moleculesthus identified may now be analyzed, either in its free form (afterresynthesis by organic chemistry or after generation of thebi-functional molecule followed by cleavage of the linker that connectsthe encoded molecule and its identifier) or in itsoligonucleotide-linked form (as a bi-functional molecule). In order toanalyze the bi-functional molecule carrying the specific encodedmolecule, individual templates of the bi-functional molecules isolatedduring the screening may be cloned (by dilution and PCR in separatewells, or by cloning into vectors and propagation in e.g. E. coli), andthen amplified by PCR, to produce many copies of the template thatencoded the recovered organic molecule. When the encoding process (Stage2 templated process) is then performed, many identical copies ofbi-functional molecules carrying the specific encoded molecule isgenerated The characteristics of the specific encoded molecule, whenlinked to its identifier template, may then be examined. Example assaysused for the analysis of the encoded molecules (in their free form orattached to identifiers) include:

Enzyme inhibition assaysAffinity-determination by competition assays and/or ELISACell-based receptor binding assaysCell-based activity assays, based on the interaction of the encodedmolecule with molecular targets on the surface of the cellsBiacore-measurements of molecule-ligand or surface-ligand interactionsAffinity and specificity/selectivity determination using arrays ofimmobilized targets (e.g. array of 100 immobilized phosphatases), ontowhich the specific encoded molecule (in free- oroligonucleotide-associated form) is addedAffinity and specificity/selectivity determination on many specificencoded molecules simultaneously, by immobilization of e.g. 1000different bi-functional molecules to an array of oligonucleotides,followed by addition of a specific fluorescent protein.

Example assays used for the analysis of the encoded molecules (in theirfree form) include:

CaCo2-cell-based analysis of membrane permeabilityIn vivo determination of animal toxicity, bioavailability of thecompounds, and other ADMET characteristics.Solubility of encoded molecule.Water-octanol partitioning measurementsMetabolic stability measurements.

In one embodiment, the reactive groups of a molecule fragment of each oftwo or more bi-functional molecules hybridized to the same template arereacted. Alternatively, the reactive group of a molecule fragment of onebi-functional molecule is reacted with a reactive group associated withthe template to which it is hybridized. Preferably the template is anoligonucleotide template.

In a further embodiment of the invention the number of wells in step a)is m and the number of wells in step f) is n, and may be the same ordifferent for each repetition of steps b) to d) in step f) n; and

the structure of the encoded (bifunctional) molecule is

O_(p,q)—( . . . —(O_(2,q)—Z)))—Y—(((X—R_(1,q))—R_(2,q))— . . .)—R_(p,q);

whereinX, Y and Z are components of the linker molecule, L, X being adapted forreaction with a molecule fragment, Z being adapted for reaction with anoligonucleotide and Y being a flexible linker connecting X and Z;O_(p,q) is the oligonucleotide identifier added in repetition number(p−1) of steps b) to f) in well number q;R_(p,q) is the molecule fragment added in repetition number (p−1) ofsteps b) to f) in well number q;p is an integer of at least 1;m and n are integers of at least 5, such as at least 10, preferably atleast 15, more preferably at least 20, and most preferably at least 50;andfor O_(1,q) and R_(1,q), q is in the range 1 to m, for O_(p,q) andR_(p,q) where p is greater than 1, q is in the range 1 to n.

In another embodiment of the present invention the number of wells instep a) is m and the number of wells in step f) is n, and n may be thesame or different for each repetition of steps b) to d) in step f); and

the structure of the encoded (bifunctional) molecule is

O_(p,q)— . . . —O_(2,q)—O_(1,q)—(((L-R_(1,q))—R_(2,q))— . . . )—R_(p,q);

whereinO_(p,q) is the oligonucleotide identifier added in repetition number(p−1) of steps b) to f) in well number q;R_(p,q) is the molecule fragment added in repetition number (p−1) ofsteps b) to f) in well number q;p is an integer of at least 2;m and n are integers of at least 5, such as at least 10, preferably atleast 15, more preferably at least 20, and most preferably at least 50;andfor O_(1,q) and R_(1,q), q is in the range 1 to m, for O_(p,q) andR_(p,q) where p is greater than 1, q is in the range 1 to n.

In the formula above the bond to a residue inside a parenthesis ( )signifies that the bond may be to any part of said residue, e.g.(L-R_(1,q))—R_(2,q) means that R_(2,q) may be bound to either of L andR_(1,q)).

In an equally important aspect the present invention provides a methodfor synthesizing an encoded molecule or one or more encoded moleculescomprising the steps of:

-   -   a) Dispensing aliquots of a nascent linker molecule L into each        of m reaction wells;    -   b) Dispensing into each of said m reaction wells a corresponding        aliquot of an m^(th) molecule fragment, R_(1,m) and a        corresponding aliquot of an m^(th) oligonucleotide, O_(1,m);    -   c) Combining all of the nascent bi-functional molecules from all        m reaction wells to produce an admixture of nascent        bi-functional molecules;    -   d) Optionally, Dispensing said admixture of nascent        bi-functional molecules into n reaction wells    -   e) Optionally, dispensing into each of the n reaction wells of        step d) a corresponding aliquot of an m^(th) molecule fragment,        R_(p,q), and a corresponding aliquot of an m^(th)        oligonucleotide or oligonucleotide identifier, O_(p,q);    -   f) Optionally, combining all of the nascent bi-functional        molecules from all m reaction wells in step e) to produce an        admixture of nascent bi-functional molecules;    -   g) Optionally repeating steps d) to f) one or more times;    -   h) contacting a resulting bi-functional molecule of step f)        or g) with one or more templates, said one or more templates        optionally being associated with a reactive group, under        conditions to allow for hybridization of each of the templates        to one or more of said nascent bi-functional molecule generated        in step f) or g);

i) Optionally, reacting reactive groups of a molecule fragment of two ormore nascent bi-functional molecules hybridized to the same template, orreacting the reactive group of a molecule fragment of one nascentbi-functional molecule with the reactive group associated with thetemplate to which it is hybridized;

the linker molecule L contains at least one reactive group capable ofreacting with a reactive group in the molecule fragment and at least onereactive group capable of reacting with a reactive group in theoligonucleotide;the molecule fragments each contain at least one reactive group capableof reacting with a reactive group in the linker molecule L or a reactivegroup in another molecule fragment, and the reactive groups of eachmolecule fragment may be the same or different;the oligonucleotide identifiers each contain at least one reactive groupcapable of reacting with a reactive group in the linker L or a reactivegroup in another oligonucleotide identifier, and the reactive groups ofeach oligonucleotide identifier may be the same or different;the oligonucleotide identifier added to each well in step b) and e)identifies the molecule fragment added to the same well in therespective step;the steps a) and b) as well as the steps d) and e) may be performed inany order;the steps d) and e) in step f) may also be performed in any order.

It is to be understood that the encoded molecule may be a bi-functionalmolecule with an encoded part comprising one or more molecule fragments,R, and a coding part comprising one or more oligonucleotide identifiers,O.

In steps b) and e) of the above process the molecule fragment as well asthe oligonucleotide is reacted with the linker to produce a nascentbi-functional molecule.

In step h) the said bi-functional molecule may be viewed as a carrier ora carrier molecule and as mentioned above the template in step i) ispreferably an oligonucleotide template.

Again, the process outlined above may be seen as a combination of a step1 synthesis and a step 2 synthesis, wherein the step 1 synthesiscomprises the steps a) to g) and the step 2 synthesis is carried out instep h.

In one embodiment of the method described in the preceding paragraphsthe number of wells in step a) is m and the number of wells in step f)is n, and for each repetition of steps b) to d) in step f) n may be thesame or different; and

the structure of the encoded (bifunctional) molecule is

O_(p,q)—( . . . —(O_(2,q)—(O_(1,q)—Z)))—Y—((((X—R_(1,q))—R_(2,q))— . . .. )—R_(p,q);

whereinX, Y and Z are components of the linker molecule, L, X being adapted forreaction with a molecule fragment, Z being adapted for reaction with anoligonucleotide and Y being a flexible linker connecting X and Z;O_(p,q) is the oligonucleotide identifier added in repetition number(p−1) of steps b) to f) in well number q;R_(p,q) is the molecule fragment added in repetition number (p−1) ofsteps b) to f) in well number q;p is an integer of at least 2;m and n are integers of at least 5, such as at least 10, preferably atleast 15, more preferably at least 20, and most preferably at least 50;andfor O_(1,q) and R_(1,q), q is in the range 1 to m, for O_(p,q) andR_(p,q) where p is greater than 1, q is in the range 1 to n.

In another embodiment the method n may for each repetition of steps d)to f) in step g) be the same or different; and

the structure of the encoded (bifunctional) molecule is

O_(p,q)— . . . —O_(2,q)—O_(1,q)—((((L-R_(1,q))—R_(2,q))— . . .)—R_(n,q);

whereinO_(p,q) is the oligonucleotide identifier added in repetition number(p−1) of steps b) to f) in well number q;R_(p,q) is the molecule fragment added in repetition number (p−1) ofsteps b) to f) in well number q;p is an integer of at least 1;m and n are integers of at least 5, such as at least 10, preferably atleast 15, more preferably at least 20, and most preferably at least 50;andfor O_(1,q) and R_(1,q), q is in the range 1 to m, for O_(p,q) andR_(p,q) where p is greater than 1, q is in the range 1 to n. Again, thebond to a residue inside a parenthesis ( ) signifies that the bond maybe to any part of said residue, e.g. (L-R_(1,q))—R_(2,q) means thatR_(2,q) may be bound to either of L and R_(1,q)).

In a further embodiment of the above described method, the structure ofthe nascent bi-functional molecules resulting from step b is

O_(1,q)-L-R_(1,q);

and the structure of the nascent bi-functional molecule obtained afterrepeating the process steps defined in step g) p−1 times is

O_(p,q)—( . . . )—O_(1,q)-L-R_(1,q)—( . . . )—R_(n,q);

wherein p is greater than or equal to 1.9.

It is preferred that the number number of reaction wells in step a)and/or the number of reaction wells in step f) is at least is at least2, such as at least 5, at least 10, at least 25, at least 50, at least100, at least 200, at least 500, at least 1000 at least, at least10,000, at least 100,000 at least 1,000,000, at least 10⁷, or at least10⁸. For certain applications m must be 100.000 or more, such as 10⁶,10⁷, or 10⁸ in step b and c, if the process steps d) to f) are notrepeated. If said steps are repeated, however, m may be as low as 10.000in steps b) and c)

Accordingly it may be preferred to repeat the process steps defined instep g) at least once, such as at least twice, such as at least threetimes, such as at least four times or more.

In certain embodiments unique identification of the molecule fragmentsmay be preferred. Accordingly, in these embodiments the oligonucleotideidentifier, O_(p,q), added in reaction well number q in repetitionnumber (p−1) of the steps specified in step g) uniquely identifies themolecule fragment, R_(p,q), added in reaction well number q inrepetition number (p−1) of the steps specified in g). In otherembodiments, however, identical oligonucleotide identifiers, O_(p,q),are added in two or more reaction wells in the same repetition number(p−1) of the steps specified in g).

Furthermore, yet other embodiments of the invention involves addition ofidentical molecule fragments, R_(p,q), in two or more wells in the samerepetition number (p−1) of the steps specified in g).

Also, it may be preferred to add two or more oligonucleotide identifiersto one or more reaction wells in a repetition of the steps specified instep g). Most preferably, however 1 oligonucleotide identifier is addedper well, but it should be recognised that it will be possible to add 2,3, 4, oligonucleotide identifier per well.

Furthermore the method according to the invention may involve adding twoor more molecule fragments to one reaction wells in each repetition ofthe steps specified in g). Whereas it is preferred to add 1 moleculefragment per well, it will also be possible to add 2, 3, 4, or 5molecule fragments per well.

In the method described above, each oligonucleotide identifier comprisesa sequence of from 2 to 100, such as from 2 to 90, from 2 to 80, from 2to 70, from 2 to 60, from 2 to 50, from 2 to 40 from 2 to 30, from 2 to25, from 2 to 20, from 2 to 15, from 2 to 10 or from 2 to 5 nucleotides,such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25,30, 35 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100nucleotides.

It should be recognised that, preferably, the number of identifiers ineach resulting bi-functional molecule is 2. However, each bi-functionalmolecule may also comprise 1, 3, 4, or 5 identifiers.

It should likewise be recognised that each template is preferablycapable of hybridising with 2 carriers. Whereas this is preferred, thetemplate may also be capable of hybridising with 1, 3, 4, 5, 6 or 7carrier molecules.

It will for most applications be preferred that the number ofnucleotides in each oligonucleotide identifier is 10-15. Other preferrednumbers of nucleotides are 5-10, 5-15, 5-20, 10-30.

It should be recognised that the said one or more templates may be anamplifiable template as well as a template, which is non-amplifiable bypolymerises. The one or more templates may thus comprise a sequenceselected from the group consisting of: nucleotides, unnaturalnucleotides, PNA, morpholinos, LNA, RNA, DNA, and other nucleotideanalogs capable of base pairing with a natural oligonucleotide orunnatural oligonucleotide. Most preferably the template comprises a DNAsequence or an RNA sequence.

In preferred embodiments of the invention said one or more templateshave a length of at least 40 nucleotides, such as 30-50 nucleotides,20-60 nucleotides, 15-80 nucleotides if not considering the primerannealing sites. When these sites are included the total length ispreferably approximately 80 nucleotides, such as 70-120 nucleotides,60-100 nucleotides, 55-120 nucleotides 50-150 nucleotides or 60-175nucleotides. In some embodiments of the invention the said one or moretemplates may even comprise up to 250 nucleotides or even up to 500nucleotides.

As for the nature of the oligonucleotide sequences each oligonucleotideidentifier and/or a sequence of two or more of the oligonucleotideidentifiers may comprise a sequence of oligonucleotides, thecomplementary sequence of which is at least 20% identical, such as atleast 30%, such as at least 40%, such as at least 50%, such as at least55%, such as at least 60%, such as at least 65%, such as at least 70%,such as at least 75%, such as at least 80%, such as at least 85%, suchas at least 90% or such as at least 95% identical to the part of saidtemplate that hybridises to the identifier. It is to be understood thatthe templates when used in the method according to the present inventionmay be comprised of coding regions separated by spacer regions.

In some preferred embodiments of the present invention the linker, L, isselected from the group consisting of flexible linkers such asPolyethylen glycol, polypeptide, polysaccharide, oligonucleotide, C₈,C₆, C1₂. In further preferred embodiments of the invention the linker,L, is a cleavable linker. For example the linker may be cleavable bybase, acid, light, reagent, heat)

In further applications of the method according to the invention themethod may involve using the bi-functional product molecule resultingdirectly from the process steps presented above, that is steps a) to h),as a carrier molecule in a second round of step 2 synthesis.Accordingly, the method may also comprise a further step comprisingcontacting the bi-functional molecule resulting from step h) with one ormore nascent bi-functional molecules and one or more templates eachcapable of recognizing at least two of the oligonucleotide identifierspresent in the bi-functional molecule. The nascent bi-functional aregenerated through steps a) through f) and the optional steps, g)). It isfurther to be understood that the identifiers may be covalently ornon-covalently linked to each other, and that the identifiers may bedouble- or single-stranded identifier oligonucleotides, with overhang orblunt-ended.

In a preferred embodiment two or more oligonucleotide identifiers may becovalently linked together, optionally in the presence of a ligase orisomerase enzyme. Alternatively two or more oligonucleotide identifiersare linked by templated extension by enzymes (polymerases).

In some embodiments of the invention two or more oligonucleotideidentifiers are ligated by chemical ligation or by a combination ofenzymatic and chemical ligation. By a combination of enzymatic andchemical ligation one may understand that two or more oligonucleotideidentifiers are ligated by enzymatic ligation in one process step andchemical ligation in a preceding or subsequent process step. Forchemical ligation of the oligonucleotide identifiers the followingreactions and pairs of reactive groups are the preferred ones:

reaction reactive group reacts w/ reactive group phophodiester 3′-OHImidazole-activated formation 5′-phosphate acylation amine esteracylation amine carboxylic acid disulfide formation SH pyridyl-disulfidereductive amination amine aldehyde phosphodiester 3′-OH pyrophosphate-formation activated 5′-phosphatetosyl displacement reaction

It is to be understood that the linker of at least one bi-functionalmolecule may be cleaved simultaneously with or subsequently tohybridisation of the oligonucleotide identifier of said nascentbi-functional molecule to the template.

In the method according to the invention said reactive groups ofmolecule fragments of the bi-functional molecules or of moleculefragments of the bi-functional molecules and of the template may bereacted in a reaction selected from the group consisting of: acylation,reductive amination, alkylhalide alkylation, Wittig reaction,sulphonoylation, isocyanate addition, Suzuki coupling, nucleophilicaromatic substitution, thiourea bond formation, carbamate formation,Heck coupling, HWE reaction, 1,3-dipolar cycloaddition, Michaeladdition, nitro aldol condensation.

In some embodiments of the invention the resulting encoded molecule hasa linear structure and is selected from the group consisting of: dimers,trimers, tetramers, pentamers, multimers, and polymers.

In further preferred embodiments at least one molecule fragment havingmore than one reactive groups has been used in the preparation of theresulting encoded molecule.

It is to be understood that the number of reactive groups pr moleculefragment may be 1, 2, 3, 4, 5 or 6. It is further to be understood that,in the final product, the reactive groups of said at least one moleculefragment has reacted with other molecule fragments. The resultingmolecule is thus a branched or scaffolded structure selected from thegroup of molecules comprising a scaffold with two substituents,scaffolds with three substituents, scaffolds with four substituents, andscaffolds with five substituents. In this context the term “substituent”means a molecule fragment that has been reacted at the substituentposition of the scaffold.

In another main aspect the invention provides a method for identifying amolecule with desired characteristics, said method comprisingsynthesizing a library of encoded molecules by a method as describedabove.

In some embodiments the method further comprises a step of subjectingthe library to a partitioning or enrichment procedure, to identifyencoded molecules with desired characteristics. In still otherembodiments the method further comprises screening a plurality ofencoded molecules in a process to identify and optionally increase therelative amount of an encoded molecule having one or more desiredcharacteristics. Various means and methods for carrying out enrichmentprocedures and screening are described in the present specificationwhich may be applied to the process.

In some preferred embodiments the method comprises identifying theencoded molecule by determining the oligonucleotide sequence(s) of theattached identifiers.

In yet another main aspect of the invention a library of encodedmolecules or bi-functional molecules is obtained. Said encode orbi-functional molecules may be obtained or may be obtainable by a methodaccording to any of claims.

In the library the number of different compounds or compound species maybe at least 100, such as at least 1000, at least 10,1000, at least100,000, at least 106, at least 10⁷, at least 10⁸, at least 10⁹, atleast 10¹⁰, at least 10¹¹, at least 10¹², at least 10¹³ or at least10¹⁴.

Additional aspects and embodiments of the invention are described inbrief in the following:

a) A method for synthesising one or more encoded molecules, comprisingthe following steps:

step 1: dispensing aliquots of a nascent linker molecule L comprisingthe components X, Y, and Z, where X is adapted for reaction with amolecule fragment, Z is adapted for reaction with an oligonucleotide andY is a flexible linker connecting X and Z, into each of m reactionwells; thenstep 2: dispensing into each of the m reaction wells of said step 1 acorresponding aliquot of the m^(th) molecule fragment R_(1,m) and acorresponding aliquot of the m^(th) oligonucleotide identifier O_(1,m)to allow reaction between the molecule fragment and X of the linker, andreaction between the oligonucleotide and Z of the linker, to produce aproduct bi-functional molecule R_(1,m)-L-O_(1,m) where the producedbi-functional molecule comprises a reactive group; thenstep 3: combining all of the nascent bi-functional molecules from all mreaction wells produced in said step 2 for producing an admixture ofnascent bi-functional molecules; thenstep 4: dispensing equal aliquots of the admixture of nascentbi-functional molecules from the prior step into each of m reactionwells, thenstep 5: dispensing into each of the m reaction wells of said step 4 acorresponding aliquot of the m^(th) molecule fragment represented byR_(n, m) and a corresponding aliquot of the m^(th) identifier moleculerepresented by O_(n, m) for producing a nascent bi-functional moleculerepresented by:

R_(n,m)—( . . . )—R_(1,m)-L-O_(1,m)—( . . . )—O_(n,m)

wherein n is greater than or equal to 2; thenstep 6: combining all of the elongated nascent bi-functional moleculesfrom all m reaction wells of step 5 for producing an admixture ofelongated nascent bi-functional molecules; thenstep 7: repeating steps 4-6 until the desired bi-functional carriermolecules, each formed from the reaction of n molecule fragments and noligonucleotide identifiers, are produced, and where each bi-functionalcarrier molecule generated comprises one or more reactive units; thenstep 8: providing one or more templates, which one or more templatesoptionally have a reactive unit associated therewith; thenstep 9: contacting one or more carrier molecules of step 7 with said oneor more templates under conditions to allow for specific hybridisationof the oligonucleotide identifiers of the one or more carrier moleculesto the one or more templates; thenstep 10: reacting the reactive units of the molecule fragments of atleast two carrier molecules hybridised to the same template, or reactingthe reactive unit of a carrier molecule with the reactive unitassociated with the template to which it is hybridised,

-   -   to generate one or more encoded molecules.

b) The method as described in a), wherein the oligonucleotideidentifiers of two or more carrier molecules are covalently linkedtogether prior to, during or after step 9 or 10.

c) The method as described in b), wherein the oligonucleotideidentifiers are covalently linked together after step 9 but before step10, by a ligase enzyme.

d) The method of b), wherein the oligonucleotide identifiers arecovalently linked together after step 9 but before step 10, in theabsence of a ligase enzyme.

e) The method of a) to d), wherein the template is dissociated from thecarrier molecules prior to reaction between said reactive units.

f) The method of a) to e), wherein more than one molecule fragment isadded to the same reaction well in steps 2 or 5, to allow for a multiplecomponent reaction to take place.

g) The method of a) to f) where m does not have the same value indifferent repetitions of step 5.

h) The method of a) to g), where in step 2 or 5 the oligonucleotideidentifiers are linked to the nascent bi-functional molecule by a ligaseenzyme.

i) The method of a) to h), where in step 2 or 5 the oligonucleotideidentifiers are linked to the nascent bi-functional molecule without theuse of a ligase enzyme.

j) The method of a) to i), wherein a library of more than onebi-functional molecule is generated, the method further comprisingenriching for library members comprising an encoded molecule displayinga desired property.

k) The methods of a) to j) wherein steps 4-7 have been eliminated.

l) The methods of a) to k), wherein steps 8-10 have been eliminated.

m) The library of two or more bi-functional molecules generated by themethod of a) to l).

n) The library of 10⁶ or more bi-functional molecules generated by themethod of a) to l).

o) The library of 10¹⁰ or more bi-functional molecules generated by themethod of a) to l).

With respect to the above description of the various aspects of thepresent invention and of the specific embodiments of these aspects itshould be understood that any feature and characteristic described ormentioned above in connection with one aspect and/or one embodiment ofan aspect of the invention also apply by analogy to any or all otheraspects and/or embodiments of the invention described.

When an object according to the present invention or one of its featuresor characteristics is referred to in singular this also refers to theobject or its features or characteristics in plural. As an example, whenreferring to “a cell” it is to be understood as referring to one or morecells.

Throughout the present specification the word “comprise”, or variationssuch as “comprises” or “comprising”, will be understood to imply theinclusion of a stated element, integer or step, or group of elements,integers or steps, but not the exclusion of any other element, integeror step, or group of elements, integers or steps.

FIGURE LEGENDS

FIG. 1. Reactions in stage 1 of the method in the invention

Schematic representation of an example of the synthesis steps ofstage 1. Two rounds of “split and mix” synthesis are shown leading tothe generation of bi-functional carrier molecules each carrying adifferent di-peptide and a unique 24-mer oligonucleotide that encodesthe di-peptide. Each round of synthesis adds an amino acid and anidentifier oligonucleotide. (m) represents the number of differentmolecule fragments in each of the two different repertoires employed.(m) can have a different values for different repertoires.

The split and mix synthesis shown in the example includes the followingsteps:

Add linker molecule to wells 1-m

Add amino acids R_(1(1-m)) to wells 1-m, and react with linker.

Add oligonucleotides O_(1(1-m)) to wells 1-m, and react with the linker.

Mix content of wells 1-m and split into 1-m wells on a new plate.

Add amino acids R_(2(1-m)) to wells 1-m and react with reactive group ofR_(1(1-m))

Add oligonulceotides O_(2(1-m)) to wells and react with O_(1(1-m)).

Mix content of wells 1-m

FIG. 2. Reactions in stage 2 of the method in the invention

Example of the synthesis steps of stage 2. In the illustrated examplethe bi-functional carrier molecules generated in the example in FIG. 1are combined by a template directed method. Tetra-peptide bi-functionalcarrier molecules with 48-mer identifier oligonucleotides are thereforegenerated in the example. (m) represents the number of differentmolecule fragments in each of the four different repertoires, i.e., inthe example m=1000 for all four repertoires. (M) represents the totalnumber of (encoded) molecules generated. Here, M=(1000)⁴=10¹².

The synthesis in the example comprise the following steps:

Add bi-functional carrier moleculefrom stage 1

Add DNA templates that bind the carriers through their complementaryoligo's

Acyl transfer reaction where the amino group of di-peptide in onecarrier attacks the peptidyl ester of the di-peptide in the othercarrier

The synthesis is complete

FIG. 3. Types of molecule fragment transfer from one carrier to another:

Direct transfer reaction: The reaction between reactive groups leadsdirectly to the transfer of a molecule fragment. The mechanism is shownschematically (“generic”) as well as for a specific case (“example”).Other types of reactions allowing direct transfer are shown in FIG. 6.

Indirect transfer reaction. Reaction between reactive groups leads tothe formation of a linkage between the two reactive groups. Thereafter amolecule fragment is cleaved off from one carrier, mediating itstransfer to another carrier. “Clv” indicates a cleavable moiety, i.e. apart of the linker that is cleavable, for example by acid, base,electromagnetic radiation, light, heat, or by specific reagents orcatalysts. Long horizontal line symbolises a template. Short horizontalline symbolises oligonucleotide identifier.

FIG. 4. Alternative methods for the reactions in stage 2

Examples of alternative methods for carrying out the reactions of stage2 are shown. A number of templates (long horizontal line) is mixed withsets of carriers (short horizontal line). In the examples, two sets ofcarriers are employed. The first example is identical to the exampleshown in FIG. 2, except that in this example the two carriers areligated together before reaction of the reactive groups of the twocarriers. The next two examples show variations of the template directedreactions illustrated in the first example. In Example 2, theidentifiers of the two carriers are ligated together prior to reactionof the reactive groups of X and Y, and the duplex structure denatured,to generate single-stranded complementary template. The single-strandedstructure improves the likelihood of X and Y reacting. The reactionefficiency of the reactive groups of X and Y may be increased byincluding complementary sequences next to X and Y. This will lead tostable duplex formation proximal to X and Y, positioning X and Y inclose proximity, and thereby increasing the reaction efficiency. InExample 3, one of the carriers is ligated to the template through itsoligonucleotide identifier moiety. The ligation of carrier to templatemay be stimulated by including a hair-pin structure in the template, asshown in the figure, and then ligating together the template and carrierby use of for example a ligase or chemical ligation. Example 4 shows atemplate-free method of carrying out the reactions in stage 2. Thecarriers of the example are double-stranded, allowing for efficientligation of their overhangs. Ligation of the carriers lead to theformation of a complementary template. Before reaction of X with Y, theduplex structure is denatured, allowing a more efficient reaction of Xand Y. As in example 3, the efficiency of reaction may be increased byincluding complementary sequences proximal to X and Y, respectively.

The library of encoded molecules that results from each of the examplescan be of the same kind; however, the examples describe differentset-ups that may allow different chemical reactions to be performed. Ina preferred embodiment, the template of the bifunctional molecules areturned into double-stranded DNA before selection or screening isperformed, in order to eliminate potential interaction from thesingle-stranded regions. Thus, in examples 2 and 4, it may beadvantageous to add a terminal oligonucleotide that anneals to the DNAtemplate that carries the encoded molecule; by extension, e.g. by apolymerase such as Sequenase, a double-stranded DNA will be generated,carrying the encoded molecule at one end.

The lower strand of the duplexes symbolises the template (longhorizontal line); short horizontal lines symbolise oligonucleotideidentifiers; after ligating the oligonucleotide identifiers together, acomplementary template is formed, symbolised by a long horizontal line.X and Y are molecule fragments, each containing at least one reactivegroup.

FIG. 5. Template generation:

An example of a DNA library generation process for the synthesis of alibrary of 10¹² DNA templates. Four sets of 1000 DNA oligos are mixedindividually with their complementary sequences, to form for example 12nt duplex DNA, with an overhang of one nucleotide at both ends. In theexample each oligo carries a region complementary to an identifiersequence (a “codon” sequence). In addition, the oligos of the two distalsets of oligos contain constant regions. These constant regions may beused at a later stage for PCR-amplification, sequencing or polymeraseextension. This is followed by a ligation step in which the overhangsmediate the ligation of the 4000 duplex DNA complexes, to form 10¹²(=1000⁴) different duplex DNA complexes. Ligation may be by a ligase orby chemical ligation. Optionally, the templates may be amplified by e.g.PCR using primers that anneal to the constant regions at the ends of thetemplate, or by any other molecular biological technique that allowsamplification. Finally, one of the strands is isolated in order to beused in a templating process (stage 2 templated synthesis). Theisolation of single stranded templates can be done in a number of ways,including asymmetric PCR on the ligated product (which leads to excessof one of the strands), or by including a biotinylated PCR-primer thatanneals to one end of the template and thus leads to incorporation ofbiotin into one of the strands of the duplex; by immobilisation of thebiotin on streptavidin-coated solid support, and denaturation of theduplex template, one may recover the non-biotinylated strand from thesupernatant, or the biotinylated strand immobilised on solid support.

In the example, the configuration of each of the 10¹² single-strandedtemplates thus is as follows: “Constantsequence-codon1-codon2-codon3-codon4-Constant sequence”. Each of thecodon positions contain one specific of the 1000 possible sequences,i.e., each template carries its specific combination of a codon1-,codon2-, codon3-, and codon4 sequence.

Templates may also be generated by stage 1 split and mix synthesis, inwhich optionally the reaction of molecule fragments with the carrier isexcluded. To generate a DNA template library as the one described inthis example, 4 sets of 1000 different duplex DNA molecules must beligated, employing 1000 wells in each of four rounds of split and mixsynthesis. This will generate the same DNA template library of 10¹²molecules as described above.

FIG. 6. Direct transfer: Reactive groups and bonds formed upon reaction:A number of reactions are shown that mediate the direct transfer ofmolecule fragments from one carrier to another. In the left part of thefigure the two carriers (the donor- and the acceptor carrier) are shown.The oligonucleotide identifiers of the carriers are indicated by a shorthorizontal line. The templates to which the carriers bind are indicatedby long horoizontal lines. The carriers carry molecule fragmentscontaining reactive groups that upon reaction lead to the transfer of amolecule fragment from one carrier onto the other. The reactions thatallow direct transfer include acylation (formation of amide, pyrazolone,isoxalone, pyrimidine, coumarine, quinolon, phtalhydrazide,diketopiperazine, hydantoin, benzodiazepinone, etc), alkylation(including reductive amination not shown in figure), vinylation,disulfide formation, addition to carbon-hetero multiple bonds, such asWittig/Wittig-Horner-Emmon (formation of substituted alkenes),transition metal catalysed reactions such as arylation (formation ofbiaryl, vinylarene), alkylation, nucleophilic substitution usingactivation of nucleophiles, such as condensations, and cycloadditions.All of these reactions may be used for indirect transfer as well. Thereactions may also be used during stage 1 synthesis. FIG. 6 is adaptedfrom (Pedersen et al. (2002) WO 02/103008 A2, “Templated molecules andmethods for using such molecules”).

FIG. 7. Indirect transfer: Reactive groups and bonds formed upon linkingreaction: Indirect transfer involves first the coupling reaction betweenthe reactive groups of carrier molecules, followed by a cleavage thatreleases one molecule fragment from its carrier molecule. This figureshows examples of reactive groups that may for example be used in thecoupling reaction. The coupling reaction may be nucleophilicsubstitution, aromatic nucleophilic substitution, transition metalcatalysed reactions, addition to carbon-carbon multiple bonds,cycloaddition to multiple bonds, and addition to carbon-hetero multiplebonds. In FIG. 8 a number of example cleavable linkers that may becombined with these coupling reactions in order to obtain efficientindirect transfer are shown. The reactions may also be used during stage1 split and mix synthesis. FIG. 7 is adapted from (Pedersen et al.(2002) WO 02/103008 A2, “Templated molecules and methods for using suchmolecules”).

FIG. 8. Cleavable linkers and protection groups, cleaving agents andcleavage products: Cleavable linkers and protection groups that may beused to release molecule fragments in an indirect transfer reaction, orcan be used as protecting groups, are shown. The cleavable linkers maybe combined with reactive groups from FIG. 7, in order to indirectlytransfer molecule fragments from one carrier to another. Linkers may becleaved by acid, base, electromagnetic radiation, light, heat, byspecific reagents or catalysts such as LiOH, PdCl₂, TCEP, NaIO₄, etc.FIG. 8 is adapted from (Pedersen et al. (2002) WO 02/103008 A2,“Templated molecules and methods for using such molecules”).

FIG. 9. A typical affinity selection process:

An example affinity selection process is shown. First a DNA templatelibrary is generated, for example as described in FIG. 5. Then, stage 2tempiated synthesis is performed using the carriers generated in stage 1(not shown), which generates a library of bi-functional molecules. Thetarget may be biotinylated, allowing its immobilisation on magneticbeads coated with streptavidin. The beads are immobilised on a magnetand washed. The bound ligands are then eluted, and the DNA of the elutedbi-functional molecules are amplified, for example by PCR, where afterthis amplified DNA can be used in yet another round of bi-functionalmolecule library synthesis, or may be sequenced in order to identify theligand structures that bound to the target.

FIG. 10. Molecular biological techniques applicable to bi-functionalmolecules:

A number of molecular biological techniques are listed that allow smallmolecule engineering, analogous to protein engineering throughmodification of the DNA encoding the protein. Using bi-functionalmolecules, one may here modify the encoded small molecule throughmodifications of the DNA encoding the small molecule. Shuffling of theDNA templates (and hence, the small molecules), can be done efficientlyby e.g. restriction endonuclease cleavage of the DNA template in thespacer that separates the codons. Other techniques such as DNA arrays ofbi-functional molecules are also suggested. FIG. 10 is modified from(Pedersen et al. (2002) WO 02/103008 A2, “Templated molecules andmethods for using such molecules”).

FIG. 11. Polyvalent display and other approaches to the identificationof molecules with weak binding characteristics.

Polyvalent display by rolling circle amplification of templates beforetemplated reaction. DNA template molecules are circularised by ligationof the ends. Specific primers are annealed and extended by rollingcircle amplification resulting in templates having multiple copies ofthe specific binding sites for carrier molecules. The multiple copytemplates are thereafter used for templated synthesis with carriermolecules resulting in polyvalent display of encoded molecules.

Stage 1 synthesis of divalent bi-functional carrier molecules.

Split and mix synthesis is carried out as in the example describingstage 1 synthesis, but the linker molecule (L) employed in the firststep of the synthesis has, in this example, two reactive ends to whichmolecule fragments (R_(1-n)) can be coupled. This results in thegeneration of divalent bi-functional carrier molecules having twoencoded molecules attached to a linker that is attached to a singleoligonucleotide identifier (O_(1-n)).

Stage 2 templated synthesis employing divalent bi-functional carriermolecules (generated in example B above).

The divalent carrier molecules from example B can be used for templatedsynthesis employing the method described for stage 2 of the presentinvention. As a result a library of divalent encoded molecules aregenerated. Each molecule consists of two encoded molecules (R_(1-n))attached to a linker that is attached to one oligonucleotide identifier(O_(1-n)).

Template assisted binding to target DNA molecule.

For screening of a library of encoded molecules for binding to targetDNA sequences, hybridisation of complementary DNA sequences (C and C′)on the bi-functional molecule and the target DNA, can increase theoverall affinity and help in the identification of molecules in thelibrary with low affinity for the target DNA.

Use of known ligand for assisted target binding.

A library of encoded divalent bi-functional molecules, each containing aknown ligand (L) and an encoded molecular entity, (R), is used forscreening for molecules with two binding sites, of which one of these isspecific for the known ligand. Binding of the known ligand to its siteon the target molecule (T), assists the binding of the encoded molecularentity to the other binding site.

Use of known ligand for assisted target binding—hybridisation of knownligand to template.

This example uses the same principle as illustrated above in FIG. 11 F,but in this example the known ligand is hybridised to the bi-functionalmolecule through hybridisation of complementary DNA sequences (C′ and C)carried by the known ligand and the template DNA. Hybridisation of theknown ligand to the template DNA of the bi-functional molecule createsfunctionally divalent molecules that can be used for screening fortarget with two binding sites, of which one is specific for the knownligand.

Use of known ligand for assisted target binding—binding to the samesite. As in the example illustrated in FIG. 11 E, but in this examplethe known ligand (L) and the molecular entity (R) bind to the same siteof the target molecule.

Use of complementary DNA attached to the target molecule to assistbinding. A DNA sequence, C′, which is attached to the target molecule,is complementary to a DNA sequence, C, on the template DNA.Hybridisation of the complementary DNA sequences assists binding of theencoded molecules to the target (T).

FIG. 12. Example set-ups allowing improved ligation of identifiers.

During the stage 1 synthesis, the identifiers are ligated together. Inorder to make this an efficient reaction, the identifiers can bedouble-stranded DNA with overhangs that are complementary, and thereforebring the reactive groups of the identifiers into close proximity.Alternatively, the identifiers are single-stranded and a complementaryoligonucleotide, or some other kind of molecule that binds to theidentifiers and brings the reactive groups into proximity, is added inorder to increase the efficiency of the chemical or enzymatic ligation.

-   -   Ligation assisted by “sticky” ends of the DNA

Ligation assisted by complementary oligonucleotide

Ligation assisted by complementary oligonucleotide attached to solidsupport

Ligation assisted by annealing to self-complementary sequence

Ligation assisted by DNA binding molecule

FIG. 13. Example molecule fragments and the encoded molecules resultingfrom stage 1 and stage 2 synthesis.

A1-A4 show generic structures of molecule fragments, carrying at least 1reactive group (A1), two reactive groups (A2), three reactive groups(A3), and four reactive groups (A4). R can be any molecular entity, andcan be cyclic or non-cyclic, aliphatic or aromatic. X, Y and A arereactive groups. Molecule fragments can carry multiple reactive groupsof the same kind (e.g., three X reactive groups), or can carry multiplereactive groups of different kinds (e.g., X, Y and A).

B1-B4 show specific examples of molecule fragments. B1 structures carryat least one reactive group (here: carboxylic acid or amine). B2structures carry at least two reactive groups (hydroxyl, amine, thiol).B3 structures carry at least three reactive groups (amine, disulfide,carboxylic acid). B4 carry at least four reactive groups (hydroxyl,amine, thiol, carboxylic acid).

C, D, E, and F show examples of molecules generated by stage 1 and/orstage 2 synthesis, i.e., by covalently coupling molecule fragmentsthrough their reactive groups. The stipled circles indicate moleculefragments that have been linked together during the stage 1 and/or stage2 synthesis.

During stage 1 synthesis the molecule fragments become attached to thelinker molecule L via reaction of a reactive group of the moleculefragment with a reactive group of the linker. In this example, thehydroxyl of the encoded molecule of (F) could have been attached to acarboxylic acid-modified oligonucleotide, thus linking the encodedmolecule to the linker.

FIG. 14. Dynamic combinatorial library of dimers or trimers of encodedmolecules.

A library, A, of encoded bi-functional molecules carries, in addition toits oligonucleotide identifier, O, an oligonucleotide sequence, C, thatis complementary to a corresponding oligonucleotide sequence carried byanother library, B, of encoded bi-functional molecules. The twolibraries are hybridised, thus creating functionally divalentbi-functional molecules that can be used in screening for targets withtwo binding sites. If appropriately designed, trimers may be formedinstead of dimers, thus creating a library of functionally trivalentencoded molecules.

FIG. 15. Molecule fragments used in example 1.

A). Molecule fragments employed in example X1 are shown.

B). List of the molecule fragments used at positions 0, 1, 2, and 3 inthe library generation process of example X1.

EXAMPLES Example 1 Formation of a Library of Bifunctional Molecules andAffinity Selection Against the Protein Target Integrin alphaV/Beta3Receptor, Employing Subprocesses 3, 5, A and (FirstSynthesis-Selection-Amplification Round), and A and i (SecondSynthesis-Selection-Round) (See Above), Using Amine Acylations for theCoupling of Molecule Fragments to Generate the Encoded Molecules

The human integrin receptor a_(v)/(3w is implicated in many biologicalfunctions such as inflammatory responses and thrombus formation as wellas cellular migration and metastatic dissemination. The natural ligandsfor alphaV/beta3 integrin receptor contain an RGD tri-peptide consensusmotif that interacts with the receptor binding pocket Consequently, muchmedical research have focused on the synthesis and identification ofsmall molecule RGD-mimetics with increased affinity for the alphaV/beta3receptor. One mimetic, Feuston 5 (Feuston et al., J Med. Chem. 2002 Dec.19; 45(26):5640-8.), comprising an arginine bioisostere coupled to a GE)dipeptide exhibits a ten-fold increased affinity (K_(D)=111 nM) comparedto the RGD-tripeptide.

It would therefore be of interest to synthesize libraries ofbifunctional molecules that include the molecule fragments that generatethe Feuston 5 ligand. In the following protocols for the generation andscreening of such libraries are described. First, the formation andscreening of a 625-membered library is described.

Stage 1 Synthesis: Generation of Two Sets of Carriers, Using ChemicalLigation and Enzymatic Ligation, Respectively, During Stage 1 Synthesisto Generate Carrier Molecules (Subprocesses 3 and 5).

FIG. 15 shows the molecule fragments and oligonucleotides employed togenerate the library.

Formation of Carrier Molecules, Set I:

Five 14 nt oligonucleotides, each containing a 5′-terminal amino-group(Glen Research catalog #10-1905-90) linked by a Spacer-PEG18 (GlenResearch catalog #10-1918-90) are synthesised by standardphosphoramidite chemistry, to give the following oligonucleotides:

O-0.1: 5′-NH2-PEG-ATGCTCGAGACGCG-3′ O-0.2: 5′-NH2-PEG-TAGCTGTAGGCGCG-3′O-0.3: 5′-NH2-PEG-AGAGCTCTGACGCG-3′ O-0.4: 5′-NH2-PEG-CGTCGTCGTACGCG-3′O-0.5: 5′-NH2-PEG-ATCGTCGAGACGCG-3′

The sequences of these oligonucleotides are not crucial, and thesequences can be changed to increase the sequence dissimilarity ordecrease the differences in annealing temperature. Each of the O-0.noligonucleotides (position 0 in the library) are now portioned out intoseparate wells (i.e., each oligonucleotide is placed in a separate well,here an eppendorf tube), and loaded with a specific molecule fragment,each of which comprises a carboxylic acid and a penteneoyl-protectedamine. The five molecule fragments are shown in FIG. 15; one of thesemolecule fragments is penteneoyl-Asp(OMe)-OH (aspartic acid, where theside chain carboxylic acid has been protected with a methyl ester). Thefollowing molecule fragment loading protocol, Protocol A, is used:

1 nmol amino-modified oligonucleotide is lyophilized and then dissolvedin 20 microliter of 100 mM Na-borate buffer, pH 8.0 with 90 mMsulpho-N-Hydroxysuccinimide (sNHS, Merck). The molecule fragments arepreactivated by incubation of 15 microliter of 100 mM molecule fragmentin DMSO and 15 microliter of 100 mM1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC, Merck)in DMF for 30 min at 30° C. before addition to the oligonucleotidesolution. Each of the five molecule fragments are added to a specificoligonucleotide, as described in FIG. 15. Following incubation for 45min at 30° C., an additional 30 microliter of pre-activated moleculefragment is added and the solution incubated for another 45 min at 30°C. Excess molecule fragment, activation agents, solvents and salt isremoved by double gel filtration using Bio-rad microspin columns 6 andeluted in MS-grade H₂O. Loading is optionally verified byElectrospray-MS analysis. Subsequently, the amino-protection group isremoved by addition of 0.2 volumes of 25 mM iodine in a mixture ofTHF/H₂O (1:1) and incubated at 37° C. for 2 h. Excess iodine is quenchedby 20 mM 2-mercaptoethanol before gel filtration purification usingBio-rad 6 microspin columns. From MS-analysis the efficiency of loadingand deprotection can optionally be estimated. At the end of this firstround of synthesis, each of the 5 oligonucleotides should be attached totheir specific molecule fragment; the molecule fragment contains areactive group, the amine, ready for reaction with the next moleculefragment that is added. The contents of the five wells are pooled, andredistributed into five new wells.

Next, 1.2 nmoles unit identifier oligonucleotides, corresponding toposition 1, are added to each well, according to the scheme of FIG. 15.The five oligonucleotides carry an imidazole-activated 5′-phosphate(Visscher, J., Schwarz, A. W. Journal of Molecular Evolution (1988), 28:3-6; Zhao, Y., Thorson, J. S. Journal of Organic Chemistry (1998),63:7568-7572) and have the following sequences:

O-1.1: 5′-ImP-CACAAGTACGAACG-3′ O-1.2: 5′-ImP-CACATAGTCTCCTC-3′ O-1.3:5′-ImP-CACATACATCGTTC-3′ O-1.4: 5′-ImP-CACATCCAGTGCAA-3′ O-1.5:5′-ImP-CACAAGCATCACTA-3′

1-2 nmoles of the oligonucleotide 3′-GCGCGTGT-5′ is added to all wells,and an appropriate buffer of pH 8-10 is added, to a final volume of20-50 microliter. This oligo is complementary to the ends ofoligonucleotides O-0.n and O-1.n, and by hybridization of thesecomplementary sequences the 3′-OH group of the oligonucleotides O-0.nand the Imidazole activated 5′-phosphates of the O-1.n oligonucleotideswill be juxtaposed. The solution is incubated for 1-5 hrs at 37° C. or50° C. This results in ligation of the juxtaposed oligonucleotides, byformation of a phosphodiester bond.

Optionally, the five solutions containing the ligation products arepurified individually using Biorad 6 spin columns according tomanufacturer's instructions and lyophilized. Next, a specific moleculefragment is reacted with each of the five solutions of nascentbifunctional molecules, according to the scheme shown in FIG. 18, usingloading protocol A described above. Excess free reactant, reagents andbuffer is removed by gelfiltration. The eluates are pooled, lyophilizedand resuspended in 40 ul of H₂O before addition of 10 ul of 25 mM iodine(in THF/H₂O, ratio 1:1) for deprotection. The reaction is incubated at37° C. for 2 h. Excess Iodine is quenched by addition of 1 ul of 1 M2-mercaptoethanol and left at ambient temperature for 5 min beforepurification of the sample using spin-gelfiltration (Bio-rad 6). Thesolution now contains 25 carrier molecules, where 25 different carrieridentifier oligonucleotides each is attached to a specific one of 25different dimers of molecule fragments. The carriers contain a freeamino group, for reaction in the templated synthesis (see below).

Formation of Carrier Molecules, Set II:

The five 15 nt oligonucleotides, corresponding to position 2 of thelibrary:

O-2.1: 3′-SH-GAGCAGGACCACCAG-5′P O-2.2: 3′-SH-CTCGACCACTACCAG-5′P O-2.3:3′-SH-CGTGCTTCCTACCAG-5′P O-2.4: 3′-SH-CCTGGTGTCGACCAG-5′P O-2.5:3′-SH-CTCGACGAGGACCAG-5′Peach carrying a 3′-terminal thiol-group, linked to the oligonucleotidethrough a flexible linker, and a 5′-terminal phosphate group, and eachportioned out into one of five separate wells, are each linked through athioester bond to a specific one of the five molecule fragments listedin FIG. 15 by the following Protocol B (Bruick et al., (1996). Currentbiology 3:49-56):

Five N-protected molecule fragments (see FIG. 15) carrying a freecarboxylic acid are first converted by standard procedures to thecorresponding thioacids. After lyophilization, 1.2 equivalents ofEllmanns Reagent (5,5′-dithiobis(2-nitrobenzoic acid)) is incubated withthe thioacid at pH 6.5 for 1 h, to produce the corresponding5-thio-2-nitrobenzoic acid ester. Optionally, the desired compounds arepurified and characterized by HPLC and mass spectrometry.

1 nmol of each of the five oligonucleotides O-2.n are now incubated inseparate wells with an excess of one of the five 5-thio-2-nitrobenzoicacid esters, according to the scheme of FIG. 15, at 25° C. or 37° C., atpH 8 for 1-5 h. Optionally, 2 mM spermidine may be added to improve theefficiency of the reaction. Optionally, the formation of the correctoligonucleotide-thioester-molecule fragment product can be verified bymass spectrometry. Finally, the five modified oligonucleotides arepooled.

Excess molecule fragment, activation agents, solvents and salt isremoved by double gel filtration using Bio-rad microspin columns 6 andeluted in MS-grade H₂O. Subsequently, the amino-protection group isremoved by addition of 0.2 volumes of 25 mM iodine in a mixture ofTHF/H₂O (1:1) and incubated at 37° C. for 2 h. Excess iodine is quenchedby 20 mM 2-mercaptoethanol before gel filtration purification usingBio-rad 6 microspin columns. Alternatively, the oligonucleotides areprecipitated with ethanol to remove the iodine. From MS-analysis theefficiency of loading and deprotection can optionally be estimated. Atthe end of this first round of synthesis, each of the 5 oligonucleotidesshould be attached to their specific molecule fragment through athioester bond; the molecule fragment contains a free amine, ready forreaction with the next molecule fragment that is added. The contents ofthe five wells are pooled, and redistributed into five new wells.

Next, 1.2 nmoles unit identifier oligonucleotides, corresponding toposition 3, are added to the wells, according to the scheme of FIG. 15.The five oligonucleotides have the following sequences:

O-3.1: 3′-CCTTAGTACGAACG-5′ O-3.2: 3′-CCTTACACGGAAAG-5′ O-3.3:3′-CCTTGCTACTAGCT-5′ O-3.4: 3′-CCTTGGAATTCCGA-5′ O-3.5:3′-CCTTGTACCATGGA-5′

1-2 nmoles of the oligonucleotide 5-TGGTCGGAA-3′, complementary to theends of oligonucleotides O-2.n and O-3.n, is added to all wells. Then,the oligos are ligated in a volume of 20 ul using ligation buffer [30 mMTris-HCl (pH 7.9), 10 mM MgCl₂, 10 mM DTT, 1 mM ATP] and 10 units T4-DNAligase at ambient temperature for 1 hour. Subsequently, the 5 solutionsof ligation products are purified individually using Biorad 6 spincolumns, and the oligonucleotides lyophilized.

Next, a specific molecule fragment is reacted with the nascentbifunctional molecule according to the scheme shown in FIG. 18 usingloading protocol A described above. Excess free molecule fragment,reagents and buffer are then removed by gelfiltration. The eluate ispooled, lyophilized and resuspended in 40 ul H₂O.

The BB-F3 molecule fragment does not react efficiently using protocol A,due to poor solubility of BB-F3 in organic solvent. Consequently, BB-F3is reacted using Protocol C instead: The ligated and lyophilized sampleis dissolved in 35 microliter 100 mM Na-borate buffer (pH 8.0) beforeaddition of 10 microliter 100 mM BB-F3 in water and 5 microliter of 500mM 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4 methylmorpholinium chloride(DMT-MM, carboxylic acid activator) and incubated at 25° C. for 2 h.Following the coupling reaction, excess molecule fragment, reagent andsalt is removed by gelfiltration as described in protocol A.

The two solutions are pooled, and the resulting solution now contains 25carrier molecules, all of which contain a thioester bond linking themolecule fragments to the carrier identifiers.

Stage 2 synthesis: Generation of bifunctional molecules by DNA-templatedsynthesis as described by (Bruick et al., (1996), Current biology3:49-56) (subprocess A, see above). Single-stranded DNA template librarygeneration. First, four sets of duplex DNAs with overhangs are produced,by standard oligonucleotide synthesis followed by hybridization ofappropriate oligonucleotide pairs, corresponding to the four encodedpositions in the library. Each set of duplex DNA in this librarycontains five different dsDNAs, corresponding to the 5 differentidentifier sequences at each position, encoding 5 different moleculefragments at each positions. All dsDNA 0.n carry a biotin as indicatedbelow for the dsDNA 0.1. When employing more molecule fragments, thenumber of dsDNAs in each set must be increased accordingly. An exampleof dsDNAs is shown below for the O-0.1, O-1.1, O-2.1, and O-3.1identifiers:

dsDNA 0.1:  5′-ATGCTCGAGACGCG-3′ 3′-GTACGAGCTCT-5′ dsDNA 1.1:    5′-CACAAGTACGAACGTATGCGTTGGCCAAACACTG-3′3′-GCGCGTGTTCATGCTTGCATACGCAACCGGXTTGTGAC-5′ dsDNA 2.1:    5′-GACCACCAGGACGAGC-3′ 3′-AAGGCTGGTGGTCCTGCTC-5′ dsDNA 3.1:5′-TTGGTTGACTAGAGACGAGCGCAAGCATGATTCC-3′3′-AACCAACTGATCTCTGCTCGCGTTCGTACT-5′

Underlined sequences are priming sites for PCR amplification. All5′-ends are phosphorylated (contain phosphates). Overhang sequences canbe extended in order to allow more efficient ligation in the preparationof the templates described below. The X in dsDNA 1.1 denotes a T thatcarries a biotin group.

The four sets of dsDNAs are incubated, to allow for hybridizationbetween overhang, and ligated as a mixture using ligation buffer [30 mMTris-HCl (pH 7.9), 10 mM MgCI₂, 10 mM DTT, 1 mM ATP] and T4-DNA ligaseat ambient temperature for 1 hour. Thus, a total of 5×5×5×5=625templates are generated. As an example, we have aligned the four dsDNAscorresponding to the O-0.1, O-1.1, O-2.1, and O-3.1 identifiersimmediately below; open spaces highlight the complementary overhangsthat hybridize during the ligation reaction:

     25            dsDNA 3.1               dsDNA 2.1       dsDNA 0.1            dsDNA 1.1-TTGGTTGACTAGAGACGAGCGCAAGCATGATTCC  GACCACCAGGACGAGC ATGCTCGAGACGCG  CACAAGTACGAACGTATGCGTTGGCCAAACACTG-3-AACCAACTGATCTCTGCTCGCGTTCGTACT  AAGGCTGGTGGTCCTGCTC GTACGAGCTCT  GCGCGTGTTCATGCTTGCATACGCAACCGGTTTGTGAC-5

The ligation product (the template) that results from the ligation ofthe above sequences is indicated immediately below:

                 dsDNA 3.1            dsDNA 2.1       dsDNA 0.1           dsDNA 1.15′-TTGGTTGACTAGAGACGAGCGCAAGCATGATTCCGACCACCAGGACGAGCATGCTCGAGACGCGCACAAGTACGAACGTATGCGTTGGCCAAACACTG-3′3′-AACCAACTGATCTCTGCTCGCGTTCGTACTAAGGCTGGTGGTCCTGCTCGTACGAGCTCTGCGCGTGTTCATGCTTGCATACGCAACCGGXTTGTGAC-5′

Thus, the sequence of identifier sequences in the 625 templates is:(primer annealing site)-O-3-O-2-O-0-O-1-(primer annealing site).Optionally, more copies of the templates can be produced by PCRamplification using primers that anneal to underlined sequences. One ofthe primers should carry a T with biotin, as indicated. Thus, theligation product and the PCR product contains a biotinylated lowerstrand.

The biotinylated double stranded product is now incubated withstreptavidin-coated beads, and the upper strand removed by alkalinedenaturation of the strands, and the pH is neutralized with anappropriate buffer to produce immobilized, single stranded template.

Hybridisation of carriers and template, and templated reaction betweenreactive groups (Bruick et al., (1996), Current biology 3:49-56): 1-100pmol immobilized templates are mixed with an excess of Set I and Set IIcarriers obtained above, in an appropriate buffer at pH 8. Optionally,the temperature is kept at 50° C. for 2 min., then lowered to 37° C. Thetemperature is now kept at 37° C. for 24 h. The carrier molecules annealto the template under these conditions; the close proximity of the aminogroup of carriers of Set I, and the thioester of carriers of Set II,leads to amide formation, in effect transferring the molecule fragmentof the thioester carrier onto the amine carrier. At this point, 625different bifunctional molecules have been generated.

Optionally, a DNA loop that can be ligated to the carrier molecule withthe 0.n and 1.n identifiers (to the right in the figure above) and tothe template, and thus covalently attaches the carrier molecule to thetemplate, can be added. Thus, optionally, to the bifunctional moleculethat results from the templated synthesis immediately above, add anoligonucleotide with the sequence5′-TATGCGTTGGCCAAACACTGGCAGATA-GAGGTCTGC-3′, where the stem sequencesare underlined, and where the 5′-terminus is phosphorylated (carries aphosphate). Add ligation buffer [30 mM Tris-HCl (pH 7.9), 10 mM MgCl₂,10 mM DTT, 1 mM ATP] and T4-DNA ligase at ambient temperature for 1hour, to covalently attach the right-ward carrier molecule (carrying theencoded molecule that results from the templated synthesis) to thetemplate.

Optional amine and carboxylic acid deprotection. Optionally, to thesolution of the previous step is now added 0.2 volumes of 25 mM iodine(in THF/H₂O, ratio 1:1) for deprotection of the penteneoyl-protectedamines. Excess Iodine is quenched by addition of 1 ul of 1 M2-mercaptoethanol and left at ambient temperature for 5 min beforeoptional purification of the sample using spin-gelfiltration (Bio-rad6). Then, optionally, NaOH is added to 25 mM, at 80° C. for 5 minutes,to deprotect methylester-protected carboxylic acids. Then increase pH to12.5 for one minOptionally, the sample is purified usingspin-gelfiltration (Bio-rad 6).

Selection on immobilised target (subprocess i).

Immobilisation and selection: Maxisorp ELISA wells (NUNC A/S, Denmark)is coated with each 100 microliter 2 ug/mL integrin alphaV/beta3(Sachem) in PBS buffer [2.8 mM NaH₂PO₄, 7.2 mM Na₂HPO₄, 0.15 M NaCl, pH7.2] overnight at 4° C. Then the integrin solution is substituted for200 microliter blocking buffer [TBS, 0.05% Tween 20 (Sigma P-9416), 1%bovine serum albumin (Sigma A-7030), 1 mM MnCI₂] and incubated for 1hour at room temperature. Then the wells are washed 2 times with 250microliter blocking buffer, and 200 microliter blocking buffer,containing the library of bifunctional molecules generated above, isadded to the wells. Following 2 hours incubation at room temperature thewells are washed with 20×250 microliter blocking buffer. After the finalwash the wells are cleared with washing buffer and the boundbifunctional molecules eluted with MeOH, glycine pH 5, or an appropriatebuffer of pH 11-13. The pH is adjusted to 7. The eluted fractioncontains potential integrin alphaV/beta3 receptor ligands.

PCR amplification of the DNA templates of the isolated bifunctionalmolecules, and cloning and characterization: The templates of the elutedfraction is now amplified by PCR, and then either cloned and sequencedfor characterization, or is taken through one more round ofsingle-stranded template preparation, and stage 2 synthesis. Forcharacterization, 5 ul eluted bifunctional molecules are used for PCR ina 25 ul reaction using 10u1 Eppendorf hotmastermix 2.5× and 10 pmol eachof forward and backwards primers that anneal to the underlined sequencesdepicted above. The PCR product is then ligated into suitable plasmidand transformed into e.g. E. coli, whereafter individual clones aresequenced by standard means (see for example below). From the DNAsequences the identity of the recovered encoded molecules can bededuced.

Template amplification, single-stranded template preparation, stage 2synthesis (e.g. subprocess 5) and selection (e.g. subprocess i). Insteadof amplifying the recovered identifiers from the selection step above,and cloning and sequencing, the bifunctional molecules can be amplifiedand taken through one more round of selection. To this end, amplify therecovered identifiers with forwards and backwards primers, where thebackwards primer carries a biotin (as indicated above). Isolatesingle-stranded DNA-template, add carriers generated above, and performstage 2 synthesis as indicated above. Finally, the selection isperformed, as indicated above, or by any other means that lead toidentification of integrin alphaV/beta3 ligands. Finally, theidentifiers recovered are PCR amplified, cloned, and sequenced (see forexample below), to reveal the identity of the encoded moleculesresponsible for binding to the integrin receptor.

Identification and characterisation.

To obtain the sequences of the DNA templates, and thereby deduce thechemical structure of the encoded molecules, the double strandedPCR-product is cloned into e.g. an E. coli vector, propagated in E.coli, and individual clones sequenced. Each of the clones represent anidentifier sequence of a bifunctional molecule in the pool isolated bythe selections; from the sequence of the DNA the corresponding encodedmolecule (that was attached to the identifier of the same bifunctionalmolecule) can be deduced. The TOPO-TA (Invitrogen Cat#K4575-J10)ligation is reacted with 4 ul PCR product, 1 ul salt solution, 1 ulvector. The reaction is incubated at RT for 30 min. Heat-shock competentTOP10 E. coli cells are thawed and put on ice. 5 ul ligation reaction isadded. Following 30 min on ice, the cells are heat-shocked at 42° C.water for 30 sec, then put on ice. 250 ul SOC is added and the cellsincubated 1 h at 37° C., before spreading on LB-ampicillin platesfollowed by incubation ON at 37° C. Individual E. coli clones are pickedand transferred to PCR wells containing 50 ul water. Colonies areincubated at 94° C. for 5 minutes and 20 ul is used in a 25 ul PCRreaction with 5 pmol of each TOPO primer M13 forward & M13 reverse andReady-To-Go PCR beads (Amersham) using the following PCR program: 94° C.2 min, then 30×(94° C. 4 sec, 50° C. 30 sec, 72° C. 1 min) then 72° C.10 min. Primers and free nucleotides are degraded by adding 1 ul EXO/SAPmixture 1:1 to 2 ul PCR product. Incubation is at 37° C. for 15 min andthen 80° C. for 15 min. 5 pmol T7 primer is added and water to 12 ul.Subsequently, 8 ul DYE-namic ET cycle sequencing Terminator Mix is addedfollowed by PCR-cycling using 30 rounds of (95° C. 20 sec, 50° C. 15sec, 60° C. 1 min). Purification is done using seq96 spinplates(Amersham), followed by analysis on a MegaBace sequenzer.

To verify that the isolated encoded molecules indeed represent ligandsto the target protein (integrin alphaV/beta3), individual bifunctionalmolecules may be prepared, by preparation of single stranded DNA of thatbifunctional molecule, and performing the templated synthesis, togenerate multiple copies of that specific bifunctional molecule. Theability of the bifunctional molecule (and, expectably, the ability ofthe encoded molecule) to bind the protein target (integrin alphaV/beta3)is then tested by e.g. immobilising the protein target in the well of amicrotiter plate, adding the bifunctional molecule, washing off unboundbifunctional molecule, and then determine the amount of boundbifunctional molecule.

Alternatively, the identified encoded molecule may be synthesized in itsfree form, by standard chemical synthesis protocols, and then examinedin e.g. competition binding experiments.

The directionality of the oligonucleotides used in the example may bechanged, so as for example to include a thiol at the 5′-end rather thanthe 3′-end, or the sequences of the oligonucleotides may be changed inorder to obtain highest possible mismatch (“sequence difference”) amongthe different unit identifiers and carrier identifiers, while keepingthe annealing temperatures relatively similar. This will increase thefidelity of the hybridization of carriers to the template during stage 2synthesis, and will also increase the fidelity of the deconvolutionstep, since sequencing errors will be less of a problem if theidentifiers have fewer identical nucleotide positions.

In the example a thioester was employed as the reactive group of Set IIcarriers. The activated ester can be any other type of activated ester(e.g., N-hydroxide succinimide ester, nitrophenyl-ester,nitrobenzyl-ester), or the ester may be a regular carboxyester. Theseactivated esters are prepared by standard organic synthesis methods.

In the example, only the Set I carriers contain a long, flexible PEGlinker. It may be advantageous that both carrier sets contain a PEGlinker, to obtain high flexibility of the molecule fragments that mustreact.

In the example, the order of reactions between molecule fragments, andligation of identifiers during stage 1 synthesis, is“reaction-ligation-reaction”. This order can be changed, to bereaction-reaction-ligation, if desired.

The constant regions of the unit identifier oligonucleotides are 4 or 5nt in the example. The constant regions are complementary to the thirdoligonucleotide added; the third oligonucleotide brings the two unitidentifiers into close proximity, and thus mediates the ligation of theunit identifiers. The overlap region between the identifier and thethird oligonucleotide can be extended (to allow for a more efficientligation during stage 1 synthesis), or shortened (to allow for morespecific annealing of the carrier molecule during the stage 2 synthesisthat follows; annealing is more specific because the sequence similaritywith other carriers employed during the stage 2 templated synthesis willbe smaller when the constant regions are shorter.

The recovered sequences from the selection experiment of example X1 willcontain an abundance of the identifier sequences encoding the moleculefragments BB98, BB99, and BB-F3, as these are the molecule fragmentsthat generate the known integrin alphaV/beta3 receptor ligand, Feuston5.

The stage 1 synthesis protocol, stage 2 synthesis protocol, screeningprotocol, and characterization protocol, can be employed as modularunits, as long as each of the four protocols are finalized by apurification to remove salts, reagents, unreacted molecule fragments,and the like. Often, an appropriate purification is spin-gelfiltration(Bio-rad 6); in order to obtain very efficient purification, twospin-gel filtrations may be performed. The following examples describeprotocols for individual stage 1 synthesis, stage 2 synthesis,screening/selection, and characterization. As mentioned, these may becombined in any desired way, as long as each of the protocols arefinalized with an appropriate purification step. Obviously, the lengthand composition of the identifiers must be designed so as to mediatespecific and efficient annealing of the carriers to the template duringtemplated synthesis.

Example 2 Formation of Five Different Libraries of BifunctionalMolecules, i.e., Libraries Containing 16, 1.6×10⁵, 6.25×10⁸, 10⁸, or10¹² Bi-Functional Molecules and Affinity Selection Against the ProteinTarget Integrin alphaV/Beta3 Receptor, Employing Subprocesses 3), 5), A)and i), Using Amine Acylations for the Coupling of Molecule Fragments toGenerate the Encoded Molecules

This example describes the generation of libraries of five differentlibraries, i.e, libraries of 16, 1.6×10⁵, 6.25×10⁶, 10⁸, or 10¹²bi-functional molecules, and the use of these libraries for selectionagainst the integrin alphaV/beta3 receptor.

The protocol described in example X1 is followed, except that the setsof molecule fragments are now changed so as to include 2, 20, 50, 100,or 1000 molecule fragments at each of the four positions, leading to theformation of libraries of 2×2×2×2=16, 20x20×20×20=1.6×10⁵,50×50×50×50=6.25×10⁶, 100×100×100×100=10⁸, or 1000×1000×1000×1000=10¹²bifunctional molecules. The molecule fragments carry the sameN-protecting group (N-penteneoyl) and a free carboxylic acid, whereforethe protocol described in example X1 can be used, except that anappropriate number of wells are used, corresponding to the number ofmolecule fragments. A number of unit identifier oligonucleotides areused that correspond to the number of molecule fragments. Because of thesize of these libraries, novel ligands not strongly related to theFeuston 5 ligand, will be identified from the bigger libraries. This isparticularly true for the libraries of 10⁸ or 10¹² bifunctionalmolecules. For library sizes larger than 10⁸ encoded molecules, ligandswill be identified that do not contain all three molecule fragmentsBB98, BB99, and BBF₃, yet have dissociation constants lower than 100micromolar.

Example 3 Covalent Attachment of a Carrier to the Template Employed inthe Stage 2 Synthesis

The structure of the identifier template of the bifunctional moleculegenerated by stage 2 synthesis, and employed in the selections, can bevaried. For example, before, during or after the templated reaction, oneof the carriers may be ligated to the template by a DNA ligase, if thetemplate for example loops back on itself, as described in FIG. 4,example 3. Optionally, an extension reaction involving a primer thatanneals to the other end of the template may be performed, in order togenerate a duplex DNA where the encoded molecule is displayed at the endof the dsDNA. This may be done by annealing 1 nmol of a primer that iscomplementary to the end of the template that is not looping back onitself, and adding sequence buffer containing 200 micromolardeoxy-ribonucleotides (dNTP) in a total volume of 100 microliter beforeaddition of 20 units of sequence and incubation at 30° C. for 1 h.Following extension the reaction mixture is used in the selection stepwithout further purification.

Example 4 Disulfide Formation During Stage 1 Synthesis, Employed toAttach a Scaffold Molecule Fragment Comprising Three Reactive Groups

This is an example of a reaction that attaches a molecule fragment toanother molecule fragment, or to the linker molecule L, throughformation of a disulfide bond (FreskgArd et al., WO 2004/039825 A2,example 1, p. 106-108). The protocol may be used in stage 1 synthesis.Similar reaction conditions can be employed in a stage 2 synthesis.

An amino-modifier C6 5′-labeled oligo (5′-X-CGTAACGACTGAATGACGT-3′),wherein X may be obtained from Glen research, cat. #10-1039-90) wasloaded with a peptide (Cys-Phe-Phe-Lys-Lys-Lys, CFFKKK) using SPDPactivation (see below). The SPDP-activation of amino-oligo was performedusing 160 uL of 10 nmol oligo in 100 mM Hepes-KOH, pH=7.5, and 40 uL 20mM SPDP and incubation for 2 h at 30° C. The activated amino-oligo wasextracted 3 times with 500 uL EtOAc, dried for 10 min in a speedvac andpurified using micro bio-spin column equilibrated with 100 mM Hepes-KOH.The loading of peptide was then performed by adding 10 uL of 100 mMattachment entity and incubating overnight at 30° C. The loadedidentifier oligo was precipitated with 2 M NH₄OAc and 2 volume 96%ethanol for 15 min at 80° C. and then centrifuged for 15 min at 4° C.and 15.000 g. The pellet was re-suspended in water and the precipitationwas repeated. Wash of the oligo-pellet was done by adding 100 uL of 70%ethanol and then briefly centrifuged. The oligo was re-dissolved in 50uL H₂O and analysed by MS. After incubation the resin was removed bycentrifugation and 15 uL of the supernatant was mixed with 7 uL ofwater, 2 uL of piperidine and imidazole (each 625 mM) and 24 uLacetonitrile. The sample was analysed using a mass spectroscopyinstrument (Bruker Daltonics, Esquire 3000plus). The observed mass was7244.93 Da, which correspond well with the calculated mass, 7244.00 Da.This experimental data exemplify the possibility to load a moleculefragment onto oligonucleotides through the formation of a disulfidebond. This particular molecule fragment (peptide) harbours threereactive groups, i.e. the amine groups of the lysine side chains, andtherefore represents a scaffold with the ability to be reacted with one,two, or three other molecule fragments that are capable of reacting withthe amine groups (e.g. carboxylic acids).

Example 5 Stage 1 Acylation Reaction

This is an example of a stage 1 acylation reaction that attaches amolecule fragment to another molecule fragment coupled to anoligonucleotide, or to a reactive group of an oligonucleotide. Similarconditions can be applied for a stage 2 acylation reaction, except thatthe incoming molecule fragment must be at high concentration, e.g.10-100 mM. The experiment is described in (Freskgård et al., WO2004/039825 A2, p. 129-137).

EDC-Based Acylation Protocol:

10 uL triethanolamine (TEA) (0.1 M in DMF) was mixed with 10 uL moleculefragment (here called “Building Block (BB)”). The building blocks thatwere tested all carry a carboxylic acid and a Pent-4-enal amineprotecting group; the concentration of the building block was 0.1 MinDMSO. From this mixture 6.7 uL was taken and mixed with 3.3 uL EDC[1-Ethyl-3-(3-Dimethylaminopropyl) carbodiimide Hydrochloride J (0.1 Min DMF) and incubated 30 minutes at 25° C. 10 uL of the Buildingblock-EDC-TEA mixture was added to 10 uL of an amino modifiedoligonucleotide (here termed “amino oligo”) (in 0.1 M HEPES buffer((4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid, SIGMA), pH 7.5 andincubated for 30 minutes. During this half hour, another 6.7 uL ofBB-TEA mix was mixed with 3.3 uL EDC (0.1M in DMF) and incubated for 30minutes at 25° C. 10 uL of this second BB-EDC-TEA mixture was then addedto the amino oligo mixture together with 10 uL of 0.1 M HEPES buffer tomaintain a 1:1 ratio of DMSO/DMF:H₂O. Then the mixture was incubated for30 minutes. During this half hour, another 6.7 uL of BB-TEA mix wasmixed with 3.3 uL EDC (0.1 M in DMF) and incubate for 30 minutes at 25°C. 10 uL of this third BB-EDC-TEA mixture was then added to the aminooligo mixture together with 10 uL of 0.1 M HEPES buffer to maintain a1:1 ratio of DMSO/DMF:H₂O. Then the mixture was incubated for 30minutes. The oligonucleotide, linked to the molecule fragment (heretermed “loaded oligo”) was then purified by gel filtration with columns(Biospin P-6, Bio-Rad) equilibrated with water. The pent-4-enal amineprotection group was then removed by addition of 0.25 volumes 25 mM I₂in 1:1 water:tetrahydrofuran (THF) and incubation at 37° C. for 2 hours.The mixture was then purified by gel filtration with spin columns(Biospin P-6, BioRad) equilibrated with water. Loaded oligos wereanalyzed by ES-MS. Molecule fragments tested included aliphatic as wellas aromatic compounds, and all were attached efficiently through amidebond formation, as evidenced by mass spectrometric data within a fewDaltons of the expected mass. See (Freskgård et al., WO 2004/039825 A2,p. 129-137).

DMT-MM-Based Acylation Protocol:

10-15 nmol of carrier oligo 2 was lyophilized and redissolved in 27.5 ulH₂O. To this was added 7.5 ul 1 M HEPES pH 7.5, 10 ul of2-amino-pent-4-enal protected (allyl-glycine) building block (0.1 M indimethyl sulfoxide), and 5 ul DMT-MM[4-(4,6-dimethoxy-1,3,5-thiazin-2-yl)-4-methylmorpholinium chloride](0.5 M in water). The mixture was incubated 4-16 hours at 25-30° C. Theoligo was purified by gel filtration (Biospin P-6, BioRad). To convertthe methyl ester moiety of the building block to a carboxylic acid, 5 ul0.4 M NaOH was added and the mixture was incubated 20 min at 80° C. Themixture was then neutralized by adding 10 ul 0.5 M HEPES pH 7.5 and 5 ul0.4 M HCI. The loaded building block oligo was purified by gelfiltration Biospin P-6, BioRad) and analyzed by ES-MS. Aliphatic as wellas aromatic building blocks were attached to the amine modifiedoligonucleotide efficiently, as evidenced by the MS-data which showedgood correlation between expected and observed mass. See (Freskgård etal., WO 2004/039825 A2, p. 129-137).

Example 6 Stage 1 Enzymatic Ligation of Oligonucleotides CarryingMolecule Fragments

This is an example of a stage 1 enzymatic ligation that attaches oneoligonucleotide, carrying a molecule fragment, to anotheroligonucleotide through covalent phosphodiester bond formation. Theexperiment is described in (Freskgård et al., WO 2004/039825 A2, p.137-143).

500 pmol loaded carrier oligo (oligonucleotide carrying a moleculefragment), 5′-phosphorylated, was mixed with 750 pmol anti-codon oligo(not carrying any molecule fragment) and 750 pmol splint oligo(comprising complementary sequences to both the carrier oligo and theanti-codon oligo). See figure immediately below, showing an example pairof carrier oligo and anti-codon oligo, as well as the splint oligo. Notethat the anti-codon oligo comprises Inosines (allowing annealing toseveral different bases, here C and A. The mixture was lyophilized andredissolved in 15 ul water. Oligos were annealed by heating and slowlycooling to 20° C. 15 ul TaKaRa ligase mixture (Takara Bio Inc) was addedand the reaction was incubated at 20° C. for 1 hour. The mixture waspurified by gel filtration (Biospin P-6, BioRad) and the efficiency ofthe ligation was checked by running an aliquot on a Novex TBE-UREA gel(Invitrogen). Both oligonucleotides carrying aliphatic and aromaticcompounds were tested; different sequences around the ligation point wasexamined as well. All oligonucleotides tested were ligated with morethan 95% efficiency. See (Freskgård et at., WO 2004/039825 A2, p.137-143).

Loaded carrier Oligo          Anti-codon oligo3′-2GGAGTCGACACATAGCTCGCp CGTCGIIIIIGCAGCCAATAGTCGT-X                 TCGAGCG--GCAGCCA                    Splint oligo

Example X7

Stage 1 synthesis of a 484-member library of bifunctional molecules(subprocess 4), and selection by affinity selection on immobilizedtarget (subprocess i).

This is an example of a stage 1 synthesis employing subprocess 4. Threerounds of encoding are employed, involving 4, 11 and 11 moleculefragments, thus generating a total of 484 bifunctional molecules thatmay be used as carriers in a templated stage 2 synthesis. The threerounds of encoding involve acylation reactions. (Freskgård et al., WO2004/039825 A2, p. 143-148).

First Encoding Round.

2 pmol of loaded identifier oligo 1.1 (i.e, a particular moleculefragment attached to the identifier oligonucleotide) was combined with20 pmol of each loaded identifier oligo 1.2, 1.3, and 1.4. (602 pmolloaded identifier oligos in total). These were mixed with 0.7 pmolbuilding block oligo 3.1.3. (i.e., a particular molecule fragmentattached to an oligonucleotide, capable of hybridizing with theidentifier oligonucleotide), and 72.7 pmol each of 10 different otherfirst round building block oligos (eg. 3.1.1 and 3.1.2; 727 pmol loadedbuilding block oligos in total). The oligos were lyophilized andredissolved in 50 ul extension buffer (EX) [20 mM HEPES, 150 mM NaCl, 8mM MgCl₂]. The mixture was heated to 80° C. and slowly cooled to 20° C.to allow efficient annealing of identifier and building block oligos. 5ul of 0.5 M DMT-MM in water was added and the mixture was incubated at37° C. for 4 hours. Extension of the identifier oligo on the buildingblock oligo identifier was performed by adding 3 ul of a 10 mM mixtureof each deoxynucleotide triphosphate [dATP, dGTP, dCTP, dTTP] and 3 uLof 13 units/ul Sequenase (Amersham Biosciences). The mixture wassubsequently incubated at 30° C. overnight. Then 3 pi of 2M NaOH wasadded and the mixture was incubated for 80° C. for 10 minutes followedby neutralization by addition of 3 pi 2M HCl. The mixture was thenpurified by passing through a gel filtration column (Biospin P-6,BioRad). 0.25 volumes of 25 mM I₂ in 1:1 THF:water was added, mixed andincubated at 37° C. for 2 hours. 60 ul binding buffer (BF) [100 mMHEPES, 150 mM NaCI] and water ad 300 ul was added. The mixture was addedto streptavidin-sepharose beads (Amersham Biosciences) pre-washed 3times in BF buffer and incubated at room temperature for 10 minutes,followed by incubation on ice for 10 minutes with gentle stirring. Thebeads were then washed three times with water. Extended identifieroligos were stripped from the building block oligos bound to thestreptaviding-sepharose beads by applying 100 ul NH3 1:1 in water andincubating at room temperature for 5 minutes.

Second Encoding Round

To the eluate was added 0.36 pmol second round loaded building blockoligo 3.2.2 and 36.4 pmol each of 10 different other second roundbuilding block oligos (eg. 3.2.1 and 3.2.3; 364 pmol loaded second roundbuilding block oligos in total) and the mixture was lyophilized andredissolved in 50 ul EX buffer. The encoding was performed essentiallyas described under above.

Final extension. The eluted identifier oligo were lyophilized anddissolved in 50 ul EX buffer. Then 200 pmol primer E38 [5′-X IIIAGATGGCAGAT-3\ X=CXS Biotin] was added. Annealing was performed byheating the mixture to 80° C. and slowly cooling to 20° C. Extension ofthe identifier oligo was performed by adding 3 ul of a 10 mM mixture ofeach deoxynucleotide triphosphate [dATP, dGTP, dCTP, dTTP] and 3 ul of13 units/ul Sequenase. The mixture was subsequently incubated at 30° C.for 2 hours. The mixture was then purified by passing through a gelfiltration column (Biospin P-6, BioRad). This eluate was used forselection. An aliquot was removed for analysis of the input in theselection procedure.

General Procedure 5: Affinity Selection on Immobilized Protein Target.

Maxisorp ELISA wells (NUNC A/S, Denmark) were coated with each 100 uL 2ug/mL integrin α_(v)β₃ (Bachem) in PBS buffer [2.8 mM NaH₂PO₄, 7.2 mMNa₂HPO₄, 0.15 M NaCl, pH 7.2] overnight at 4° C. Then the integrinsolution was substituted for 200 pi blocking buffer [TBS, 0.05% Tween 20(Sigma P-9416), 1% bovine serum albumin (Sigma A-7030), 1 mM MnCl₂]which was left on for 3 hours at room temperature. Then the wells werewashed 10 times with blocking buffer and the encoded library was addedto the wells after diluting it 100 times with blocking buffer. Following2 hours incubation at room temperature the wells were washed 10 timeswith blocking buffer. After the final wash the wells were cleared ofwash buffer and subsequently inverted and exposed to UV light at 300-350nm for 30 seconds. Then 100 ul blocking buffer without Tween-20 wasimmediately added to each well, the wells were shaken for 30 seconds,and the solutions containing eluted identifiers were removed for PCRanalysis.

Analysis of Selection Input and Output.

PCR was performed on the input and output of the selection, usingprimers corresponding to the 5′ end of the identifier oligos and the E38primer. PCR was performed using Ready-To-Go (RTG) PCR beads (AmershamBiosciences) and 10 pmol each primer in a reaction volume of 25 ul. ThePCR reaction consisted of an initial denaturation step of 94° C. for 2minutes followed by 30-45 cycles of denaturation at 94° C. for 30seconds, annealing at 58° C. for 1 minute and extension at 72° C. for 1minute. A final extension step of 2 minutes at 72° C. was included. ThePCR products were resolved by agarose gel electrophoresis and the bandcorresponding to the expected size was cut from the gel and purifiedusing QIAquick Gel Extraction Kit (QIAGEN). To sequence individual PCRfragments the purified PCR products were cloned into the pCR4-TOPOvector (Invitrogen) according to the manufacturer's instructions. Theresulting mixture was used for transformation of TOP10 E. coli cells(Invitrogen) using standard procedures. The cells were plated on growthmedium containing 100 ug/ml ampicillin and left at 37° C. for 12-16hours. Individual E. coli clones were picked and transferred to PCRwells containing 50 ul water. These wells were then boiled for 5 minutesand 20 ul mixture from each well was used in a PCR reaction using RTGPCR beads and 5 pmol each of M13 forward and reverse primers accordingto the manufacturer's instructions. A sample of each PCR product wasthen treated with Exonuclease I (USB) and Shrimp Alkaline Phosphatase(USB) to remove degrade single stranded DNA and dNTPs and sequencedusing the DYEnamic ET cycle sequencing kit (Amersham Biosciences)according to the manufacturer's instructions and the reactions wereanalyzed on a MegaBace 4000 capillary sequencer (Amersham Biosciences).Sequence outputs were analyzed with ContigExpress software (InformaxInc.). A overview of molecule fragments used for library generation isshown in (Freskgård et al., WO 2004/039825 A2, p. 146-147).

Theoretically, the integrin a_(v)p₃ ligand A (Molecule 7 in Feuston B.P. et al., Journal of Medicinal Chemistry 2002, 45, 5640-5648) ispresent in 1 out of 3×10⁸ bifunctional molecules in this library. Thecodon combination compatible with encoding of ligand A was not found in28 sequences derived from the encoded library before selection (input)in agreement with the expected low abundance of this codon combination(1 in 3×10⁸). A codon combination compatible with encoding of ligand Awas found in 5 out of 19 sequences derived from the encoded libraryafter selection in integrin aVB3-coated wells. These numbers thuscorrespond to an apparent enrichment factor of (3×10⁸/(19/7))=8×10⁷.

For more detailed date see (Freskgård et al., WO 2004/039825 A2, p.143-148).

Example 8 Selection of Bifunctional Molecules Using Size-ExclusionChromatography

This is an example of subprocess iii), although a real library ofbifunctional molecules are not screened. A protocol for selectionemploying size-exclusion chromatography is presented. The experiment istaken from (FreskOrd et al., WO 2004/039825 A2, p. 148-150).

This example illustrates the possibility to use column separation toperform selection on complexes against various targets. In this example,size-exclusion chromatography (SEC) is used, but other types ofchromatography can be used where target-bound complexes are separatedfrom the non-bound complexes. The complex is exemplified in this exampleby a biotin molecule attached to an oligonucleotide sequence with apredetermined sequence (see below). Thus, the nucleotide sequence of theidentifier specifies the identity of the synthetic molecule as biotin.The encoding sequence can have any length and be divided into discreteregions for encoding various building blocks as discussed elsewhereherein. Also, the displayed molecule can have a linear or scaffoldstructure. Biotin-AATTCCGGAACATACTAGTCAACATGA Biotin is known to bind tostreptavidin. The binding of biotin to streptavidin will link theidentifier to the target molecule and therefore change the identifiersphysical and chemical properties, such as e.g. the apparent molecularweight. This change is possible to detect using e.g. size-exclusionchromatography: 78 pmol of the complex molecule was loaded on a Superdex200, PC 3.2/30 column (AKTA-FPLC, AmershamPharmaciaBiotech) and analysedin PBS buffer with a flow rate of 0.050 ml/min. As may be seen from thespectrogram, the complex molecules retention-time was approximately 35minutes. When the target (83 pmol streptavidin) was analysed underidentical conditions the retention-time was approximately the same. Thelow absorption of the target molecules is due to the wavelength (260 nm)used in the measurement. At this wavelength, the extinction coefficientis high for the nucleotides in the complexes but low for the proteintarget.

However, when the complex molecules was premixed with the targetmolecules (78 pmol complex and 83 pmol target incubated for about 1 h inPBS buffer) to allow binding and then analysed under identicalconditions, the retention-time change significantly (28 minutes). Thechange is due to the increase in molecular weight (or hydrodynamicvolume) due to the binding of the complex to the target. This will allowthe separation of the target-bound complexes from the non-boundcomplexes. The fraction that contains the complexes and the targetmolecules are pooled and amplified using appropriate primers. Theamplified identifiers can then be used to decode the structures of theenriched displayed molecules. The strategy of performingcolumn-selection of libraries of bifunctional complexes has two majoradvantages. First, the enriched (target-bound) complexes are elutedbefore the non-bound complexes, which will drastically reduce thebackground from the non-bounded complexes. Secondly, the enrichment onthe column will be extensive due to all the separation steps in thepores in the matrix. The separation of the target-bound complexes usingthis approach will be dependend on the molecular weight of the complexesbut predominantly of the molecular weight of the target. The molecularweight of the target can be adjusted by linking the target to a supportthat increases the apparent molecular weight. The increased molecularweight will enhance the separation by reducing the retention-time on thecolumn. This can be done using for example a fusion protein, antibody,beads, or cross-linking the target in multimeric form. Thus, the targetprotein can be expressed as a fusion protein or a specific antibody canbe use to increase the molecular weight. The target can be immobilizedon small beads that permit separation and the target can be cross-linkedusing standard reagents to form multimers or cross-linked to a carriermolecule, for example another protein. Preferably, the molecular weightis increased so the target molecules elute in the void volume of thecolumn. Examples of other types of column separation that can be usedare affinity chromatography, hydrophobic interaction chromatography(HIC), and ion-exchange chro-matography. Examples of column media, otherthan Superdex, that can be used in size-exclusion chromatography are:Sephacryl, Sepharose or Sephadex.

Example 9 Encoded Multiple Component Reaction (MCR) During a Stage 1Synthesis

This is an example of a stage 1 synthesis that involves the reaction ofmultiple different encoded molecule fragments in the same well; this isan example of an UGI reaction. The experiment is described in (Freskgårdet al., WO 2004/039825 A2, p. 157-162). Preparation ofaldehyde-comprising scaffold-oligo, using 4-carboxybenzaldehyde. Asolution of 4-carboxybenzaldehyde (scaffold) in DMF (25 uL, 150 mM) wasmixed with 25 uL of a 150 mM solution of EDC in DMF. The mixture wasleft for 30 min at 25° C. 50 uL aminooligo (10 nmol) in 100 mM HEPESbuffer pH 7.5 was added and the reaction mixture was left for 20 min at25° C. Excess scaffold was removed by extraction with EtOAc (500 uL) andremaining EtOAc was removed in vacuo by spinning 10 min in a speedvac.The mixture was then purified by gel filtration with spin columns(Biospin P-6, BioRad) equilibrated with water. The loaded oligo wereanalyzed by ES-MS.

Multi-component reaction. A solution of the Benzaldehyde loaded oligoprepared above (200 pmol) was lyophilized and redissolved in 10 uL H₂O.2-Methoxy ethylamine in methanol (10 uL, 40 mM), 3-furan-2-yl-acrylicacid in methanol (10 uL, 40 mM), and cyclohexyl isocyanide in methanol(10 uL, 40 mM) was added and incubated overnight at 37° C. The reactionmixture was diluted with 40 uL H₂O and purified by gel filtration withspin columns (Biospin P-6, BioRad) equilibrated with water. MCR-producton oligo was analyzed by ES-MS. The starting benzaldehyde loaded oligowas identified in the MS-spectrum together with the UGI product.

Multi-component reaction. A solution of benzaldehyde loaded oligo (320pmol) was lyophilized and redissolved in 10 uL H₂O. 2-Amino ethanol inmethanol (10 uL, 40 mM), 3-Methoxy-propionic acid in methanol (10 uL, 40mM), and ethyl isocyanoacetate in methanol (10 uL, 40 mM) was added andincubated overnight at 37° C. The reaction mixture was diluted with 40uL H₂O and purified by gel filtration with spin columns (Biospin P-6,BioRad) equilibrated with water. MCR-product on oligo was analyzed byES-MS. The starting benzaldehyde loaded oligo was identified in theMS-spectrum together with three products, Diketopiperazine, UGI productand the Amine product.

Encoding. Excess reactants, activation agents, solvents and salt wasremoved by double gel-filtration using Bio-rad microspin columns 6 andeluted in MS-grade H₂O and loading was verified by Electrospray-MS(Bruker Inc) analysis before the displayed molecule attached to theoligonucleotide was encoded. The benzaldehyde loaded oligonucleotide,that has been reacted with the other three components to form thedisplayed molecule as described above was mixed with the codonoligonucleotides L2, L3 and L4 together with the splint oligonucleotidesS1, S2 and 53 (sequences shown below) and ligated using a ligase (T4 DNAligase). The ligation was performed using the following conditions. Thedouble stranded oligonucleotide was achieved by mixing the encodingstrands (L1, L2, L3 and L4) with the splint oligonucleotides (S1, S2 andS3) to form a 7 oligonucleotide hybridisation product (for efficientannealing and ligation). About 50 pmol of each specific oligonucleotidewas used and the oligonucleotides was ligated in a volume of 20 uL usingligation buffer [30 mM Tris-HCl (pH 7.9), 10 mM MgCI₂, 10 mM DTT, 1 mMATP] and 10 units T4-DNA ligase at ambient temperature for 1 hour.

LI: 5′-CGATGGTACGTCCAGGTCGCA-3′ SI: 5′-ATCGTGCTGCGACCT-3′ L2:5′-GCACGATATGTACGATACACTGA-3′ S 2: 5′-GTGCCATTCAGTGT-3′ L3:5′-ATGGCACTTAATGGTTGTAATGC-3′ S3: 5′-TGTATGCGCATTAC-3′ L4:5′-GCATACAAATCGATAATGCAC-3′

The identifier comprising the tags was amplified using a forward (FP)and reverse (RP) primer using the following conditions: 5 uL of theligated identifier oligonucleotide was used for PCR in a 25 uL reactionusing 10 uL Eppendorph hotmastermix 2.5× and 10 pmol each of AH361 &Frw-27. PCR was run: (ENRICH30): 94° C. 2 min, then 30 cycles of [94° C.30 sec, 58° C. 1 min, 72° C. 1 min], then 72° C. 10 min.

FP: 5′-CGATGGTACGTCCAGGTCGCA-3′ RP: 5′-GTGCATTATCGATTTGTATGC-3′

The amplified identifier oligonucleotide was cloned to verify that theassembled oligonucleotides contained the codon region (CGTCC, GTACG,AATGG and TCGAT). The TOPO-TA (Invitrogen Cat#K4575-J10) ligation wasreacted with 4 ul PCR product, 1 ul salt solution, 1 ul vector. Thereaction was incubated at RT for 30 min. Heat-shock competent TOP10 E.coli cells was thawed and put on ice. 5 ul ligation reaction was added.Following 30 min on ice, the cells were heat-shocked at 42° C. water for30 sec, and then put on ice. 250 ul SOC was added and the cellsincubated 1 h at 37° C., before spreading on LB-ampicillin platesfollowed by incubation ON at 37° C. Individual E. coli clones werepicked and transferred to PCR wells containing 50 uL water. Colonieswere incubated at 94° C. for 5 minutes and 20 uL was used in a 25 uL PCRreaction with 5 pmol of each TOPO primer M13 forward & M13 reverse(AH365/AH366) and Ready-To-Go PCR beads (Amersham) using

PCR program: 94° C. 2 min, then 30×(94° C. 4 sec, 50° C. 30 sec, 72° C.1 min) then 72° C. 10 min.

Primers and free nucleotides were degraded by adding 1 pi EXO/SAPmixture 1:1 to 2 uL PCR product. Incubation was at 37° C. for 15 min andthen 80° C. for 15 min. 5 pmol T7 primer (AH368) was added and water to12 uL. Subsequently, 8 uL DYE-namic ET cycle sequencing Terminator Mixwas added followed by PCR-cycling using 30 rounds of (95° C. 20 sec, 50°C. 15 sec, 60° C. 1 min). Purification was done using seq96 spinplates(Amersham), followed by analysis on a MegaBace sequenizer.

Example 10 Stage 1 “Click” Reaction

This is an example of stage 1 synthesis, using the “click” reaction.Similar conditions can be applied to stage 2 “click” reactions. Theexperiment is described in (U.S. patent application 60/588,672, p.34-35.)

General procedure. An alkyne-containing DNA conjugate is dissolved inpH8.0 phosphate buffer at a concentration of ca. 1 mM. To this mixture isadded 10 equivalents of an organic azide and 5 equivalents each ofcopper (II) sulfate, ascorbic acid, and the ligand(tris-((1-benzyltriazol-4-yl)methyl)amine) all at room temperature. Thereaction is followed by LCMS, and is usually complete after 1˜2 h. Theresulting triazole-DNA conjugate can be isolated by ethanolprecipitation.

Preparation of Azidoacetyl-Gly-Pro-Phe-Pra-NH₂: Using 0.3 mmol ofRink-amide resin, the indicated sequence was synthesized by automatedsynthesis with Fmoc-protected amino acids and HATU as activating agent(Pra=C-propargylglycine). Azidoacetic acid was used to cap thetetxapeptide. The peptide was cleaved from the resin with 20% TFA/DCMfor 4 h. Purification by RP HPLC afforded product as a white solid (75mg, 51%). NMR (DMSO-d₆, 400 MHz): 8.4-7.8 (m, 3H), 7.4-7.1 (m, 7H),4.6-4.4 (m, 1H), 4.4-4.2 (m, 2H), 4.0-3.9 (m, 2H), 3.74 (dd, 1H, J=6 Hz,17 Hz), 3.5-3.3 (m, 2H), 3.07 (dt, 1H, J=5 Hz, 14 Hz), 2.92 (dd, 1H, J=5Hz, 16 Hz), 2.86 (t, 1H, J=2 Hz), 2.85-2.75 (m, 1H), 2.6-2.4 (m, 2H),2.2-1.6 (m, 4H). IR (mull) 2900, 2100, 1450, 1300 cm″¹. ESIMS 497.4([M+H], 100%), 993.4 ([2M+H], 50%). ESIMS with ion-source fragmentation:519.3 ([M+Na], 100%), 491.3 (100%), 480.1 ([M−NH₂], 90%), 452.2([M−NH₂—CO], 20%), 424.2 (20%), 385.1 ([M−Pra], 50%), 357.1 ([M−Pra-00],40%), 238.0 ([M−Pra-Phe], 100%).

Cyclization of Azidoacetyl-Gly-Pro-Phe-Pra-NH2: The azidoacetyl peptide(31 mg, 0.62 mmol) was dissolved in MeCN (30 mL). Diisopropylethylamine(DIEA, 1 mL) and Cu(MeCN)JPF₆ (1 mg) were added. After stirring for 1.5h, the solution was evaporated and the resulting residue was taken up in20% MeCN/H₂O. After centrifugation to remove insoluble salts, thesolution was subjected to preparative reverse phase HPLC. The desiredcyclic peptide was isolated as a white solid (10 mg, 32%). ¹H NMR(DMSO-d₆, 400 MHz): 8.2S (t, 1H, J=5 Hz), 7.77 (s, 1H), 7.2-6.9 (m, 9H),4.98 (m, 2H), 4.48 (m, 1H), 4.28 (ra, 1H), 4.1-3.9 (m, 2H), 3.63 (dd,1H, J=5 Hz, 16 Hz), 3.33 (m, 2H), 3-0 (m, 3H), 2.48 (dd, 1H, J=11 Hz, 14Hz), 1.75 (m, 1H0, 1.55 (m, 1H), 1.32 (m, IH), 1.05 (m, 1H). IR(mull)2900, 1475, 1400 cm″¹. ESIMS 497.2 ([M+H], 100%), 993.2 ([2M+H], 30%),1015.2 ([2M+Na], 15%). ESIMS with ion-source fragmentation: 535.2 (70%),519.3 ([M+Na], 100%), 497.2 ([M+H], 80%), 480.1 ([M−NH_(2], 30)%), 452.2([M−NH₂—CO], 40%), 208.1 (60%).

Example 11 A Stage 1 Synthesis Involving Aromatic NucleophilicSubstitution

This is an example of an aromatic nucleophilic substitution reactionemployed in a stage 1 synthesis. Similar conditions may be used in stage2 synthesis. The experiments are described in (U.S. patent application60/588,672, p. 36)

General Procedure for Arylation of DNA-linker with Cyanuric Chloride:DNA-Linker is dissolved in pH 9.5 borate buffer at a concentration of 1mM. The solution is cooled to 4° C. and 20 equivalents of cyanuricchloride is then added as a 500 mM solution in MeCN. After 2 h, completereaction is confirmed by LCMS and the resulting dichlorotriazine-DNAconjugate is isolated by ethanol precipitation.

Procedure for Amine Substitution of Dichlorotriazine-DNA:Dichlorotriazine-DNA is dissolved in pH 9.5 borate buffer at aconcentration of 1 mM. At room temperature, 40 equivalents of analiphatic amine is added as a DMF solution. The reaction is followed byLCMS and is usually complete after 2 h. The resultingmonochlorotriazine-DNA conjugate is isolated by ethanol precipitation.

Procedure for Amine Substitution of Monochlorotriazine-DNA:(Alkylamino)-monochlorotriazine-DNA is dissolved in pH 9.5 borate bufferat a concentration of 1 mM. At 42° C., 40 equivalents of a secondaliphatic amine is added as a DMF solution. The reaction is followed byLCMS and is usually complete after 2 h. The resultingdiaminotriazine-DNA conjugate is isolated by ethanol precipitation.

Example 12 A Stage 1 Synthesis (Subprocess 9) and Characterization of aLibrary of 10⁵ Members

This is an example of a stage 1 synthesis, involving five synthesisrounds (here termed “cycles”), employing acylation reactions for thecoupling of molecule fragments (here termed “building blocks”). Similarconditions can be used in stage 2 synthesis. The experiments aredescribed in (U.S. patent application 60/588,672, p. 26-34). Thesynthesis of a library comprising on the order of 10⁵ distinct memberswas accomplished using the following reagents:

Compound 1: An approximately 19 by duplex DNA, where the two strands atone end has been covalently linked, and where that end includes a PEGlinker and a teminal amino group; and where the 5′-end of one strand atthe other end carries a 5′-phophate.

Building block precursors: 12 compounds, each of which contains aFmoc-protected amino group and a free carboxylic acid. The compoundsinclude aliphatic as well as aromatic compounds and aliphatic cyclicstructures.

Oligonucleotide tags: A total of 60 duplex DNAs, with 7 central basepairs and 2 nt overhangs at both ends, and 5′-phosphates at both ends,are included. The 60 duplex DNAs correspond to 5 cycles where 12 tagsare added, one per building block precursor, in each round.

IX ligase buffer: 50 mM Tris, pH 7.5; 10 mM dithiothreitol; 10 mM MgCl₂;2.5 mM ATP; 50 mM NaCl.

10× ligase buffer: 500 mM Tris, pH 7.5; 100 mM dithiothreitol; 100 mMMgCl₂; 25 mM ATP; 500 mM NaCl

Cycle 1

To each of twelve PCR tubes was added 50 uL of a 1 mM solution ofCompound 1 in water; 75 uL of a 0.80 mM solution of one of Tags1.1-1.12; 15 uL 10× ligase buffer and 10 uL deionized water. The tubeswere heated to 95° C. for 1 minute and then cooled to 16° C. over 10minutes. To each tube was added 5,000 units T4 DNA ligase (2.5 uL of a2,000,000 unit/mL solution (New England Biolabs, Cat. No. MO202)) in 50ul IX ligase buffer and the resulting solutions were incubated at 16° C.for 16 hours. Following ligation, samples were transferred to 1.5 mlEppendorf tubes and treated with 20 uL 5 M aqueous NaCl and 500 ul cold(−20° C.) ethanol, and held at −20° C. for 1 hour. Followingcentrifugation, the supernatant was removed and the pellet was washedwith 70% aqueous ethanol at −20 ^(c)C. Each of the pellets was thendissolved in 150 uL of 150 mM sodium borate buffer, pH 9.4.

Stock solutions comprising one each of building block precursors BB1 toBB12, N,N-diisopropylethanolamine and0-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate, each at a concentration of 0.25 M, were prepared insodium phosphate buffer, pH 8.0, and incubated at room temperature for20 minutes. Each solution (6 uL) was diluted with 30 uLN₅N,-dimethylformamide and added to the appropriate eppendorf tube. Twoadditional 6 uL aliquots of building block precursor stock solution wereadded after 20 minutes and 40 minutes, respectively, for a final ratioof 30:1 building block precursor to tag. The tubes were gently shakenfor 2 hours at 4° C. The tags and corresponding building blockprecursors used in Round 1 are set forth in Table 1, below.

TABLE 1 Building Block Precursor Tag BB1 1.11 BB2 1:6 BB3 1.2 BB4 1-8BB5 1.1 BB6 1.10 BB7 1.12 BB8 1.5 BB9 1.4 BB10 1.3 BB11 1.7 BB12 1.9

Following acylation, the 12 reaction mixtures were pooled and theresulting mixture was lyophilized to yield a dry residue, which wasdissolved in 1.7 mL water. Two volumes of cold 100% ethanol were addedand the mixture was allowed to stand at −20° C. for at least one hour.The mixture was then centrifuged for 15 minutes at 14,000 rpm in a 4° C.microcentrifuge. Following centrifugation, as much supernatant aspossible was removed with a 1 mL micropipet; the mixture was thencentrifuged again, and the remainder of the supernatant was removed witha 200 jiL pipet. Cold 70% ethanol (200 uL) was then added to the rube,and the mixture was centrifuged for 5 minutes at 4° C.

The supernatant was then removed with a 200 uL pipet; and the remainingethanol was allowed to evaporate at room temperature over 5 to 10minutes. The remaining pellet was suspended in 2 mL water and purifiedby HPLC with a 50 mM aqueous triethylammonium acetate mobile phase at pH7.5. The fractions containing the library were collected, pooled andlyophilized. The resulting residue was redissolved in 2.5 mL aqueousNa₂HPO4 and 100 uL piperidine was added, resulting in the formation of aprecipitate. The precipitate was separated from the supernatant bycentrifugation and washed with 200 uL water. The wash and thesupernatant were combined and used for Cycle 2.

Cycles 2-5

For each of these cycles, the combined solution resulting from theprevious cycle was divided into 12 equal aliquots of 50 ul each andplaced in PCR tubes. To each tube was added a solution comprising adifferent tag, and ligation, purification and acylation were performedas described for Cycle 1, except that for Cycles 3-5, the HPLCpurification step described for Cycle 1 was omitted. The correspondencebetween tags and building block precursors for Cycles 2-5 is presentedin Table 2.

The products of Cycle 5 were ligated with the closing primer shownbelow, using the method described above for ligation of tags.

5′-PO₃-GGCACATTGATTTGGGAGTCA GTGTAACTAAACCCTCAGT-PO₃-5′

TABLE 2 Building Block Precursor Cycle 2 Tag Cycle 3 Tag Cycle 4 TagCycle 5 Tag BB1 2.7 3.7 4.7 5.7 BB2 2.8 3.8 4.8 5.8 BB3 2.2 3.2 4.2 5.2BB4 2.10 3.10 4.10 5.10 BBS 2.1 3.1 4.1 5.1 BB6 2.12 3.12 4.12 5.12 BB72.5 3.5 4.5 5.5 BB8 2.6 3.6 4.6 5.6 BB9 2.4 3.4 4.4 5.4 BB10 23 3.3 4.35.3 BB11 2.9 3.9 4.9 5.9 BB12 2.11 3.11 4.11 5.11

Results:

The synthetic procedure described above has the capability of producinga library comprising 12^(s) (about 249,000) different structures. Thesynthesis of the library was monitored via gel electrophoresis of theproduct of each cycle. The gel electrophoresis shows that each cycleresults in the expected molecular weight increase and that the productsof each cycle are substantially homogeneous with regard to molecularweight.

Example 13 Direct Transfer Acylation Reaction

This is an example of a stage 2 synthesis direct transfer reaction,involving the reactive group NH2, and an activated ester,N-hydroxysuccinimide ester. Similar reaction conditions can be appliedto the stage 1 acylation reaction, except that the concentration of theincoming molecule fragment must be higher (e.g., 100 mM incomingmolecule fragment in a stage 1 synthesis). The example is taken from(Freskgård et al., WO 2004/039825 A2, example 3, p. 111-116.

The molecule fragment, in the following called “attachment entitiy (AE)”is in the following experiments either a scaffold molecule fragment,e.g. the peptide, CFFKKK, attached to an oligonucleotide, in thefollowing called “identifier”, or a molecule fragment, in the followingcalled “recipient reactive group” exemplified by an amino modifiedoligonucleotide. These molecule fragments allow transfer of three or onemolecule fragments, respectively. The identifier used in this experimentis an oligonucleotide coupled to the peptide CFFKKK as described inExample 4. The molecule fragment, in the following called “functionalentity (FE)”, is in this experiment 4-Pentynoic acid, which is attachedto an oligonucleotide. The identifier oligonucleotide, coupled to theCFFKKK scaffold, is annealed to the oligonucleotide carrying the4-pentynoic acid, thereby bringing the two molecule fragments into closeproximity. The annealing is directed by the complementarity of the twooligonucleotides. The annealing was performed using 600 pmol of the4-pentynoic acid oligonucleotide and 400 pmol identifier oligonucleotidein 0.1 M MES buffer at 25° C. in a shaker for 2 hours. After annealingand subsequent reaction between the two molecule fragments, the samplewas purified by micro-spin gel filtration and analyzed by MS. Theobserved mass was 7323.45 Da, which correspond well with the calculatedmass, 7324.00 Da. Thus, the MS shows a mass corresponding to thetransfer of the molecule fragment (4-pentenoic acid) onto the aminogroup of the identifier oligonucleotide through formation of an amidebond. Another example of transfer of a molecule fragment is shown belowusing the amine-modified oligonucleotide directly as the AE on theidentifier molecule. The functional entity on the building blockmolecule used in this experiment was 4-pentynoic acid. The annealing wasperformed using 500 pmol of either carrier molecule in 0.1 M MES bufferand incubating the mixture at 25° C. in a shaker for 2 hours. Themolecule fragment (4-pentenoic acid) was transferred to the amino groupon the identifier molecule during the annealing (see below). Afterannealing and transfer the sample was purified by micro-spin gelfiltration and analyzed by MS. The observed mass was 6398.04 Da, whichcorrespond well with the calculated mass, 6400.00 Da. Thus, the MSspectra of the identifier molecule after transfer of the functionalentity show a mass corresponding to the transferred molecule fragment,4-pentenoic acid, onto the identifier molecule, by formation of an amidebond. Another example of direct transfer of a molecule fragment byacylation uses the amine modified oligo directly as the identifiermolecule. The functional entity used in this experiment was Hexynoicacid. The annealing was performed using 500 pmol of either carriermolecule in 0.1 M MES buffer incubated at 25° C. in a shaker for 2hours. The hexynoic acid molecule fragment was transferred to the aminogroup on the identifier molecule through formation of an amide bond (seebelow). After annealing and transfer the sample was purified bymicro-spin gel filtration and analyzed by MS. The observed mass was6411.96 Da, which correspond well with the 15 calculated mass, 6414 Da.Thus, the MS spectra show a mass corresponding to the transfer ofhexynoic acid onto the amine of the identifier oligo through amide bondformation.

Example 14 Multi-Step Stage 2 Synthesis Using Different Types ofCleavable Linkers

This is an example of a multistep, stage 2 synthesis involving severalcarriers hybridizing to different positions of the same template, andthe use of three different types of cleavable linkers, employed inindirect transfer reactions. Also described is a templated Wittigreaction, a direct transfer reaction. The description of the experimentis taken from (Liu et al., WO 2004/016767 A2, example 3, p. 112-117).The figures referred to are from the same patent application.

Three distinct strategies have been developed to link chemical reagents(reactive units) with their decoding DNA oligonucleotides, and to purifyproduct after any DNA-templated synthetic step. When possible, an idealreagent-oligonucleotide linker for DNA-templated synthesis positions theoligonucleotide as a leaving group of the reagent. Under this“autocleaving” linker strategy, the oligonucleotide-reagent bond iscleaved as a natural chemical consequence of the reaction (see WO2004/016767 A2, FIG. 28A).

As the first example of this approach applied to DNA-templatedchemistry, a dansylated Wittig phosphorane reagent (WO 2004/016767 A2,compound (1)) was synthesized in which the decoding DNA oligonucleotidewas attached to one of the aryl phosphine groups (Hughes (1996)TETRAHEDRON LETT. 37: 7595). DNA-templated Wittig olefination withaldehyde-linked template 2 resulted in the efficient transfer of thefluorescent dansyl group from the reagent to the template to provideolefin 3 (WO 2004/016767 A2, FIG. 28A). As a second example of anautocleaving linker, DNA-linked thioester 4 (WO 2004/016767 A2), whenactivated with Ag(I) at pH 7.0 (Zhang et al. (1999) J. AM. CHEM. SOC.121: 3311) acylated amino-terminated template 5 to afford amide product6 (WO 2004/016767 A2, FIG. 28B).

Ribosomal protein biosynthesis uses aminoacylated tRNAs in a similarautocleaving linker format to mediate RNA-templated peptide bondformation. To purify desired products away from unreacted reagents andfrom cleaved oligonucleotides following DNA-templated reactions usingautocleaving linkers, biotinylated reagent oligonucleotides and washingcrude reactions with streptavidin-linked magnetic beads (see WO2004/016767 A2, FIG. 30A) were utilized. Although this approach does notseparate reacted templates from unreacted templates, unreacted templatescan be removed in subsequent DNA-templated reaction and purificationsteps.

Reagents bearing more than one functional group can be linked to theirdecoding DNA oligonucleotides through second and third linkerstrategies. In the “scarless linker” approach (WO 2004/016767 A2, FIG.28C), one functional group of the reagent is reserved for DNA-templatedbond formation, while the second functional group is used to attach alinker that can be cleaved without introducing additional unwantedchemical functionality. The DNA-templated reaction then is followed bycleavage of the linker attached through the second functional group toafford desired products (WO 2004/016767 A2, FIG. 28C). For example, aseries of aminoacylation reagents such as (D)-Phe derivative 7 (WO2004/016767 A2) were synthesized in which the alpha-amine is connectedthrough a carbamoylethylsulfone linker (Zariing et al (1980) J.IMMUNOLOGY 124: 913) to its decoding DNA oligonucleotide. The product(WO 2004/016767 A2, compound (8)) of DNA-templated amide bond formationusing this reagent and an amine-terminated template (WO 2004/016767 A2,(5)) was treated with aqueous base to effect the quantitativeelimination and spontaneous decarboxylation of the linker, affordingproduct 9 containing the cleanly transferred amino acid group (WO2004/016767 A2, FIG. 28C). This sulfone linker is stable in pH 7.5 orlower buffer at 25° C. for more than 24 hours yet undergoes quantitativecleavage when exposed to pH 11.8 buffer for 2 hours at 37 C.

In some cases it may be advantageous to introduce one or more newchemical groups as a consequence of linker cleavage. Under a thirdlinker strategy, linker cleavage generates a “useful scar” that can befunctionalized in subsequent steps (WO 2004/016767 A2, FIG. 28C). As anexample of this class of linker, amino acid reagents such as the (L)-Phederivative 10 were generated linked through 1,2-diols (Fruchart et al.(1999) TETRAHEDRON LETT. 40: 6225) to their decoding DNAoligonucleotides. Following DNA-templated amide bond formation withamine terminated template (WO 2004/016767 A2, compound (5)), this linkerwas quantitatively cleaved by oxidation with 50 mM aqueous sodiumperiodate (NaI04) at pH 5.0 to afford product 12 containing an aldehydegroup appropriate for subsequent functionalization (for example, in aDNA-templated Wittig olefination, reductive amination, or nitrolaldoladdition).

FIG. 29 of (WO 2004/016767 A2) shows the results of exemplaryDNA-templated synthesis experiments using autocleaving linkers, scarlesslinkers, and useful scar linkers. The depicted reactions were analyzedby denaturing PAGE. Lanes 1-3 were visualized using UV light without DNAstaining; lanes 4-10 were visualized by staining with ethidium bromidefollowing by UV-transillumination. Conditions for 1 to 3 were: oneequivalent each of reagent and template, 0.1 Ni TAPS buffer pH 8.5, 1 MNaCl, at 25° C. for 1.5 hours. Conditions for 4 to 6 were: threeequivalents of 4,0;I′¹ M. MES buffer pH 7.0, 1 M sodium nitrite (NaNO₂)10 mM silver nitrate (AgNC<3), at 37° C. for 8 hours. Conditions for 8to 9 were 0.1 M 3-(cyclohexylamino)-1-5-propanesulfonic acid (CAPS)buffer pH 11.8, 60 mM (3-mercaptoethanol (BME), at 37° C. for 2 hours.Finally, conditions for 11 to 12 were: 50 mM aqueous NaI04, at 25° C.for 2 hours. Ri=NH(CH₂)₂NH-dansyl; R₂=biotin.

Desired products generated from DNA-templated reactions using thestarless, or useful scar linkers can be readily purified usingbiotinylated reagent oligonucleotides (WO 2004/016767 A2, FIG. 30B).Reagent oligonucleotides together with desired products are firstcaptured on streptavidin-linked magnetic beads. Any unreacted templatebound to reagent by base pairing is removed by washing the beads withbuffer containing 4 M guanidinium chloride. Biotinylated moleculesremain bound to the streptavidin beads under these conditions. Desiredproduct then is isolated in pure form by eluting the beads with linkercleavage buffer (in the examples above, either pH 11 or sodium periodate(NaI04)-containing buffer), while reacted and unreacted reagents remainbound to the beads.

As one example of a specific library generated as described above, threeiterated cycles of DNA-templated amide formation, traceless linkercleavage, and purification with streptavidin-linked beads were used togenerate a non-natural tripeptide (WO 2004/016767 A2, FIGS. 31A-B). Each20 amino acid reagent was linked to a unique biotinylated 10-base DNAoligonucleotide through the sulfone linker described above. The 30-baseamine-terminated template programmed to direct the tripeptide synthesiscontained three consecutive 10-base regions that were complementary tothe three reagents, mimicking the strategy that would be used in amulti-step DNA-templated small molecule library synthesis.

In the first step, two equivalents of 13 (see WO 2004/016767 A2) wereactivated by treatment with 20 mM EDC, 15 mM sulfo-NHS, 0.1 M MES bufferpH 5.5, and 1 M NaCl, for 10 minutes at 25° C. The template then wasadded in 0.1 M MOPS pH 7.5, and 1M NaCl, at 25° C. and was allowed toreact for 1 hour. The free amine group in 14 (see WO 2004/016767 A2)then was elaborated in a second and third round of DNA-templated amideformation and linker cleavage to afford dipeptide 15 and tripeptide 16

(see WO 2004/016767 A2) using the following conditions: two equivalentsof reagent, 50 mM DMT-MM, 0.1 M MOPS buffer pH 7.0, 1 M NaCl, at 25° C.for 6 hours. Desired product after each step was purified by capture onavidin-linked beads and elution with 0.1 M CAPS buffer pH 11.8, 60 mMBME, at 37° C. for 2 hours. The progress of each reaction andpurification was followed by denaturing polyacrylamide gelelectrophoresis (WO 2004/016767 A2, FIG. 31B, bottom). Lanes 3, 6, and 9represent control reactions using reagents containing scrambledoligonucleotide sequences.

The progress of each reaction, purification, and sulfone linker cleavagestep was followed by denaturing polyacrylamide gel electrophoresis. Thefinal tripeptide linked to template 16 (see WO 2004/016767 A2) wasdigested with the restriction endonuclease EcoBI and the digestionfragment containing the tripeptide was characterized by MALDI massspectrometry. Beginning with 2 nmol (˜20 ug) of starting material,sufficient tripeptide product was generated to serve as the template formore than 10⁶ in vitro selections and PGR reactions (Kramer et al.(1999) CURRENT PROTOCOLS IN MOL. BIOL. 3: 15.1) (assuming 1/10,000molecules survive selection). No significant product was generated whenthe starting material template was capped with acetic anhydride, or whencontrol reagents containing sequence mismatches were used instead of thecomplementary reagents (WO 2004/016767 A2, FIG. 31B).

A non-peptidic multi-step DNA-templated small molecule synthesis thatuses all three linker strategies developed above was also performed (WO2004/016767 A2, FIG. 32A-32B). An amine-terminated 30-base template wassubjected to DNA-templated amide bond formation using an aminoacyl donorreagent (WO 2004/016767 A2, compound (17)) containing the diol linkerand a biotinylated 10-base oligonucleotide to afford amide 18 (WO2004/016767 A2) (two equivalents 17 in 20 mM EDC, 15 mM sulfo-NHS, 0.1 MMES buffer pH 5.5, 1 M NaCl, 10 minutes, 25° C., then add to template in0.1 M MOPS pH 7.5, 1M NaCl at 16° C. for 8 hours). The desired productthen was isolated by capturing the crude reaction on streptavidin beadsfollowed by cleaving the linker with NaI04 to generate aldehyde 19 (WO2004/016767 A2). The DNA-templated Wittig reaction of 19 with thebiotinylated autocleaving phosphorane reagent 20 (WO 2004/016767 A2)afforded fumaramide 21 (WO 2004/016767 A2) (three equivalents 20, 0.1 MTAPS pH 9.0, 3 M NaCl at 25° C. for 48 hours). The products from thesecond DNA-templated reaction were partially purified by washing withstreptavidin beads to remove reacted and unreacted reagent. In the thirdDNA-templated step, fumaramide 21 was subjected to a DNA-templatedconjugate addition (Gartner et al. (2001) J. AM. CHEM. SOC. 123: 6961)using thiol reagent 22 (WO 2004/016767 A2) linked through the sulfonelinker to a biotinylated oligonucleotide (three equivalents 22, 0.1 MTAPS pH 8.5, 1 M NaCl at 25° C. for 21 hours). The desired conjugateaddition product (WO 2004/016767 A2, compound (23)) was purified byimmobilization with streptavidin beads. Linker cleavage with pH 11buffer afforded final product 24 (WO 2004/016767 A2) in 5-10% overallisolated yield for the three bond forming reactions, two linker cleavagesteps, and three purifications (WO 2004/016767 A2, FIGS. 32A-32B). Thefinal product was digested with EcoRI and the mass of the smallmolecule-linked template fragment was confirmed by MALDI massspectrometry (exact mass: 2568, observed mass: 2566±5). As in thetripeptide example, each of the three reagents used during thismulti-step synthesis, annealed at a unique location on the DNA template,and control reactions with sequence mismatches yielded no product (WO2004/016767 A2, FIG. 32B, bottom). In FIG. 32B, bottom lanes 3, 6, and 9represent control reactions. As expected, control reactions in which theWittig reagent was omitted (step 2) also did not generate productfollowing the third step. Taken together, the DNA-templated syntheses ofcompounds 16 and 24 (see WO 2004/016767 A2) demonstrate the ability ofDNA to direct the sequence-programmed multi-step synthesis of botholigomeric and non-oligomeric small molecules: unrelated in structure tonucleic acids.

Example 15 Stage 2 Reactions in Organic Solvents

This is an example of a stage 2 synthesis performed in organic solvents.Similar or identical conditions can be applied to stage 1 synthesis,except that the concentrations of molecule fragments must beappropriately high to obtain efficient reaction, e.g. higherconcentrations of molecule fragments than 10 mM. The description of theexperiment is taken from (Liu et al., WO 2004/016767 A2, example 4, p.117-118). The figures referred to are from the same patent application.

A variety of DNA-templated reactions can occur in aqueous media. It hasalso been discovered that DNA-templated reactions can occur in organicsolvents, thus greatly expanding the scope of DNA-templated synthesis.Specifically, DNA templates and reagents have been complexed with longchain tetraalkylammonium cations (see, Jost et al., (1989) NUCLEIC ACIDSRES. 17:2143; Melnikov et al. (1999) LANGMUIR 15: 1923-1928) to permitquantitative dissolution of reaction components in anhydrous organicsolvents including CH2Cl2, CHCI3, DMF and methanol. Surprisingly, it wasfound that DNA-templated synthesis can indeed occur in anhydrous organicsolvents with high sequence selectivity.

FIG. 33, WO 2004/016767 A2 shows DNA-templated amide bond formationreactions where the reagents and templates are complexed withdimethyldidodecylammonium cations either in separate vessels or afterpreannealing in water, lyophilized to dryness, dissolved in CH₂Cl₂, andmixed together. Matched, but not mismatched, reactions provided productsboth when reactants were preannealed in aqueous solution and when theywere mixed for the first time in CH₂Cl₂ (WO 2004/016767 A2, FIG. 33).DNA-templated amide formation and Pd-mediated Heck coupling in anhydrousDMF also proceeded sequence-specifically.

These observations of sequence-specific DNA-templated synthesis inorganic solvents imply the presence of at least some secondary structurewithin tetraalkylammonium-complexed DNA in organic media, and shouldpermit DNA receptors and catalysts to be evolved towards stereoselectivebinding or catalytic properties in organic solvents. Specifically,DNA-templated reactions that are known to occur in aqueous media,including conjugate additions, cycloadditions, displacement reactions,and Pd-mediated couplings can also be performed in organic solvents.

It is contemplated that reactions in organic solvents may be utilizedthat are inefficient or impossible to perform in water. For example,while Ru-catalyzed olefin metathesis in water has been reported (Lynn etal. (1998) J. AM. £HEM. SOC. 120: 1627-1628; Lynn et al. (2000) J. AM.CHEM. SOC. 122: 6601-6609; Mohr et'al. (1996) ORGANOMETALLICS 15:4317-4325), the aqueous metathesis system is extremely sensitive to theidentities of the functional groups. The functional group tolerance ofRu-catalyzed olefin metathesis in organic solvents, however, issignificantly more robust. Some exemplary reactions to utilize inorganic solvents include, but are not limited to, 1,3-dipolarcycloaddition between nitrones and olefins which can proceed throughtransition states that are less polar than ground state startingmaterials.

Example 16 Stage 2 Omega Synthesis (Subprocess F), Involving AmineAcylation, Wittig Olefination, 1,3-Dipolar Cycloaddition and ReductiveAmination

This is an example of a stage 2 synthesis employing the Omega DNAarchitecture during the templated synthesis. Also described are theconditions allowing amine acylation, Wittig olefination, 1,3-DipolarCycloaddition and Reductive amination reactions to proceed efficiently.The same conditions can be applied during a stage 1 synthesis involvingthe same reactions, except that the molecule fragments must be added ata higher concentration (e.g. 10-100 mM molecule fragment). Thedescription of the experiments is taken from (Liu et al., WO 2004/016767A2, example 5, p. 118-126). The figures referred to are from the samepatent application.

This example discloses two different template architectures that furtherexpand the scope of nucleic acid-templated synthesis. During a nucleicacid-templated chemical reaction a portion of a template anneals to acomplementary sequence of an oligonucleotide-linked reagent, holdingfunctional groups on the template and transfer unit in reactiveproximity. Template architecture can have a profound effect on thenature of the resulting reaction, raising the possibility ofmanipulating reaction conditions by rationally designingtemplate-reagent complexes with different secondary structures. It washypothesized that the distance dependence of certain DNA-templatedreactions such as 1,3-dipolar cycloadditions and reductive animationcould be overcome by designing a new architecture that permits a reagentto anneal to two distinct and spatially separated regions of thetemplate. In the “Omega” architecture (see WO 2004/016767 A2, FIG. 7),the template oligonucleotide contains a small number of constant basesat, for example, the reactive 5′-end of the template in addition todistal coding regions. The oligonucleotide of the transfer unit for theOmega architecture contains at its reactive 3′-end the bases thatcomplement the constant region of the template followed by bases thatcomplement a coding region anywhere on the template. The constantregions were designed to be of insufficient length to anneal in theabsence of a complementary coding region. When the coding region of thetemplate and transfer unit are complementary and anneal, the elevatedeffective molarity of the constant regions induces their annealing.Constant region annealing forms a bulge in the otherwise double-strandedtemplate-reagent complex and places groups at the ends of the templateand reagent in reactive proximity. This design permitsdistance-dependent DNA-templated reactions to be encoded by bases distalfrom the reactive end of the template.

The efficiency of DNA-templated synthesis using the Omega architecturewas compared with that of the standard E and H architectures. The Omegaarchitectures studied comprise (i) three to five constant bases at the5′ end of the template followed by (ii) a five- to 17-base loop and(iii) a ten-base coding region. As a basis for comparison, fourdifferent classes of DNA-templated reactions were performed thatcollectively span the range of distance dependence observed to date.

Amine acylation reactions are representative of distance independentreactions that proceed efficiently even when considerable distances(e.g., 30 bases) separate the amine and carboxylate groups. As expected,amine acylation (20 mM DMT-MM, pH 7.0, at 30° C. for 12 hours) proceededefficiently (46-96% yield) in all architectures with both small andlarge distances between reactive groups on the reagent and template (WO2004/016767 A2, FIG. 34, lanes 1-5; and FIG. 35A). The Omegaarchitecture mediated efficient amine acylation with three, four, orfive constant bases at the reactive ends of the template and reagent and10 or 20 bases between annealed reactants (n=10 or 20). Importantly,control reactions in which the distal coding region contained threesequence mismatches failed to generate significant product despite thepresence of the complementary three- to five-base constant regions atthe ends of the template and reagent 5 (see WO 2004/016767 A2, FIG. 34,lane 5 for a representative example). The Omega architecture, therefore,did not impede the efficiency or sequence-specificity of thedistance-independent amine acylation reaction.

DNA-templated Wittig olefination reactions proceed at a significantlylower rate when the aldehyde and phosphorane are separated by largernumbers of template bases, eventhough product yields typically areexcellent after 12 hours or more of reaction regardless of interveningdistance. After only 2 hours of reaction (pH 7.5, 30° C.) in the E or Harchitectures, however, yields of olefin products were three- tosix-fold lower when reactants were separated by ten or more bases (n=10or 20) than when reactants are separated by only one base (n=1) (WO2004/016767 A2, FIG. 34, lanes 6-7, and FIG. 35B). In contrast, theOmega architecture with four or five constant bases at the reactive endresulted in efficient and sequence-specific Wittig product formationafter 2 hours of reaction even when 10 or 20 bases separated the codingregion and reactive end of the template (WO 2004/016767 A2, FIG. 34,lanes 8-9, and FIG. 35B). These results suggest that the constantregions at the reactive ends of the template and transfer unit in theOmega architecture permit the aldehyde and phosphorane moieties to reactat an effective concentration comparable to that achieved with theE-architecture when n=1 (WO 2004/016767 A2, FIG. 34).

Among the many DNA-templated reactions studied to date, the 1,3-dipolarcycloaddition and reductive animation reactions demonstrate the mostpronounced distance dependence. Both reactions proceed in low to modestefficiency (7%-44% yield) under standard reaction conditions using the Eor H architectures when 10 or 20 bases separate the annealed reactivegroups (WO 2004/016767 A2, FIG. 34, lanes 10-11 and 14-15, and FIGS.35C-35D). This distance dependence limits the positions on a DNAtemplate that can encode these or other similarly distant dependentreactions. In contrast, both 1,3-dipolar cycloaddition and reductiveanimation proceed efficiently (up to 97% yield) andsequence-specifically when encoded by template bases 15-25 bases awayfrom the functionalized end of the template using the Omega architecturewith four or five constant bases (WO 2004/016767 A2, FIG. 34, lanes12-13 and 16-17, and FIGS. 35C-35D). These results demonstrate that thetemplates Omega architecture permits distance-dependent reactions to beefficiently directed by DNA bases far from the reactive end of thetemplate. By overcoming the distance dependence of these reactions whilepreserving the efficiency of distant independent reactions, the Omegaarchitecture may permit virtually any contiguous subset of bases in asingle-stranded 30-base template to encode any viable DNA-templatedreaction. Interestingly, the Omega templates with only three constantbases at their reactive ends do not consistently improve the efficiencyof these reactions compared with the E-architecture (WO 2004/016767 A2,FIGS. 35C-35D), suggesting that four or five constant bases may berequired in the Omega architecture to fully realize favorable proximityeffects.

In order to probe the structural features underlying the observedproperties of the Omega architecture, the thermal denaturation of theOmega-5 and E architectures using n=10 and n=20 reagents werecharacterized. For all template-reagent combinations, only a singlecooperative melting transition was observed. Compared to the Earchitecture reagent lacking the five-base constant region, the Omega-5reagent increased the hypochromicity upon annealing by ˜50% but did notsignificantly affect melting temperature in either phosphate-bufferedsaline (PBS) or in 50 mM sodium phosphate pH 7.2 with 1 M NaCl (WO2004/016767 A2, FIG. 36). These results are consistent with a model inwhich template-reagent annealing in the Omega architecture is dominatedby coding region interactions even though the constant region formssecondary structure once the coding region is annealed. The entropiccost of partially ordering the loop between the coding and constantregions may, therefore, be offset by the favorable interactions thatarise upon annealing of the constant region.

DNA templates of arbitrary length are easy to synthesize and undesiredcross-reactivity between reactants in the same solution can be avoidedusing concentrations that are too low to allow non-complementaryreactants to react intermolecularly. These features of DNA-templatedsynthesis permit more than one DNA-templated reaction to take place on asingle template in one solution, saving the effort associated withadditional DNA-templated steps and product purifications. MultipleDNA-templated reactions per step can be difficult using the E, H, orOmega architectures, because the reagent oligonucleotide that remainsannealed to the template following the first reaction forms a relativelyrigid double helix that can prevent a second reagent annealed furtheraway along the template from encountering the reactive end of thetemplate. To overcome this, the reactive group on the template was movedfrom the end of the oligonucleotide to the middle, attaching thereactive group to the non-Watson-Crick face of a base. This “T”architecture (see WO 2004/016767 A2, FIG. 7G) was designed to permit twoDNA-templated reactions, one with a reagent coupled to the 5′ end of theoligonucleotide of a first transfer unit and one with a reagent coupledto the 3′-end of the oligonucleotide of a second transfer unit, to takeplace sequence-specifically in the same solution on a single template.

To test the viability of the T-architecture in DNA-templated reactions,the efficiency of the amine acylation, Wittig olefination, 1,3-dipolarcycloaddition, and reductive amination reactions using the Tarchitecture was studied. The T architecture sequence-specificallydirected these four reactions with efficiencies comparable to or greaterthan those of the E or H architectures (WO 2004/016767 A2, FIG. 37,69-100% yield when n=1).

It can thus be concluded that it is possible to perform each of thosereactions in an efficient way, providing high yields, at least for oneDNA architecture.

The observed degree of distance dependence using the T architecture foreach of the four reactions was consistent with the above findings (WO2004/016767 A2, compare FIG. 37 and FIG. 35). Together these resultsdemonstrate that the T architecture can mediate sequence-specific andefficient DNA-templated synthesis.

Once the ability of the T architecture to support efficientDNA-templated synthesis was established, the ability of the Tarchitecture to direct two DNA-templated reactions on one template inone solution was studied. Two different two-reaction schemes using the Tarchitecture were performed. In the first scheme, depicted in (WO2004/016767 A2, FIG. 38A), a benzaldehyde-linked T template (WO2004/016767 A2, (1)) was combined with a phosphine-linked reagent (WO2004/016767 A2, (2)) and an alpha-iodoamide-linked reagent (WO2004/016767 A2, (3)) in a single solution (pH 8.5, 1 M NaCl, at 25° C.for 1 hour). The phosphine-linked oligonucleotide complemented ten basesof the template 5′ of the aldehyde (n=−4), while the iodide-linkedoligonucleotide complemented ten bases 3′ of the aldehyde (n=0).DNA-templated SN2 reaction between the phosphine and alpha-iodoamidegenerated the corresponding phosphorane, which then participated in aDNA-templated Wittig reaction to generate cinnanamide 4 (WO 2004/016767A2) in 52% overall yield after 1 hour (FIG. 38B, lanes 9-10). Controlreactions containing sequence mismatches in either reagent generated nodetectable product. The additional control reaction lacking the aldehydegroup on the template generated only the SN2 reaction product (FIG. 38B,lanes 3-4) while control reactions lacking either the phosphine group orthe alpha-iodoamide group did not generate any detectable products (FIG.38B, lanes 5-8). In a second two-reaction scheme mediated by the Tarchitecture, depicted in (WO 2004/016767 A2, FIG. 38C), an amine-linkedT template (WO 2004/016767 A2, (5)) was combined with apropargylglycine-linked 5′.

reagent (WO 2004/016767 A2, (6)) at n=−1 and a phenyl azide-linked 3′reagent (WO 2004/016767 A2, (7)) at n=1. The addition of 20 mM DMT-MM atpH 7.0 to induce amide formation followed by the addition of 500 uMcopper(n) sulfate and sodium ascorbate to induce the recently reportedSharpless-modified Huisgen 1,3-dipolar cycloaddition provided1,4-disubstituted triazoyl alanine adduct 8 (WO 2004/016767 A2) in 32%overall yield.

Taken together, these observations show that the T architecture permitstwo sequence-specific DNA-templated reactions to take place on onetemplate in one solution. Importantly, the T architecture templatesdescribed above were accepted as efficient templates for both a singlecycle of primer extension as well as standard PCR amplification usingTaq DNA polymerase, consistent with the known tolerance of several DNApolymerases for modifications to the non-Watson-Crick face of DNAtemplates. In addition to reducing the number of separate DNA-templatedsteps needed to synthesize a target structure, this architecture mayalso permit three-component reactions commonly used to build structuralcomplexity in synthetic libraries to be performed in a DNA-templatedformat.

In summary, the Omega and T architectures significantly expand the scopeof DNA-templated synthesis. By enabling distance-dependent DNA-templatedreactions to be encoded by bases far away from the reactive end of thetemplate, the Omega architecture expands the types of reactions that canbe encoded anywhere on a DNA template. The T architecture permits twoDNA-templated reactions to take place on a single template in one step.

Materials and Methods

Oligonucleotide Synthesis. Unless otherwise specified, DNAoligonucleotides were synthesized and functionalized as previouslydescribed using 2-[2-(4-monomethoxytrityl)aminoethoxy]ethyl-(2-cyanoethyl)-N,N-diisopropyl-phosphoramidite (GlenResearch, Sterling, Va., USA) for S-functionalized oligonucleotides, andusing(2-dimethoxytrityloxymethyl-6-fluorenylmethoxycarbonylamino-hexane-1-succinoyl)-longchain alkylamino-CPG (Glen Research, Sterling, Va., USA) for3′-functionalized oligonucleotides (Calderone et al. (2002) ANGEW. CHEM.INT. ED. ENGL. 41:4104; (2002) ANGEW. CHEM. 114: 4278). In the case oftemplates for the T architecture, amine groups were added using5′-dimethoxytrityl-5-[N-(trifluoroacetylaminohexyl)-3-acrylimido]-2′-deoxyuridine-3¹-[(2-cyanoethyl)-(N,N-diisopropyl)_(j)-phosphorarnidite(Glen Research, Sterling, Va., USA) and then acylated as reportedpreviously (Calderone et al. (2002) supra).

Amine Acylation.

Amine-labeled and carboxylic acid-labeled DNA were combined in aqueous100 mM MOPS buffer, 1 M NaCl, pH 7.0 (60 nM in template DNA, 120 nM inreagent DNA) in the presence of 20 mM DMT-MM. Reactions proceeded for 12hours at 25° C.

Wittig Olefination.

Aldehyde-labeled and phosphorane-labeled DNA were combined in aqueous100 mM MOPS, 1 M NaCl, pH 7.5 (60 nM in template DNA, 120 nM in reagentDNA). Reactions proceeded for 2 hours at 30° C.

1,3-Dipolar Cycloaddition.

Dialdehyde-labeled DNA was incubated in 260 mM

N-methylhydroxylamine hydrochloride for 1 hour at room temperature(Gartner et al. (2002) J. AM. CHEM. SOC. 124: 10304). It wassubsequently combined with succinimide-labeled DNA in aqueous 50 mMMOPS, 2.8 M NaCl, pH 7.5 (final concentrations of N-methylhydroxylaminehydrochloride 0.75 mM, 60 nM in template DNA and 9.0 nM in reagent DNA).Reactions proceeded for 12 hours at 37° C.

Reductive Animation.

Amine-labeled and aldehyde-labeled DNA were combined in aqueous 100 mMMES buffer, 1 M NaCl, pH 6.0 (60 nM in template DNA, 120 nM in reagentDNA). Sodium cyanoborohydride was added as a 5 M stock in 1 M NaOH to afinal concentration of 38 mM, and reactions proceeded for 2 hours at 25°C. Reactions were quenched by ethanol precipitation in the presence of15 mM methylamine.

T Architecture-Mediated Conversion of Compound 1 to 4.

The 5′-phosphine-linked oligonucleotide (WO 2004/016767 A2, (2)) wasgenerated by coupling N-succinimidyliodoacetate (SIA) to the aminederived from 12-(4-monomethoxytritylamino)dodecyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite (Glen Research,Sterling, Va., USA) using the T (n=−4) oligonucleotide listed below,followed by treatment with 4-diphenylphosphinobenzoic acid as describedpreviously (Gartner et al. (2002) supra). The 3′-omega-iodoamide-linkedreagent (WO 2004/016767 A2, (3)) was prepared by reacting the T (n=1)oligonucleotide (see below) with SIA as described previously (Gartner etal. (2001) supra). Aldehyde-labeled template (WO 2004/016767 A2, (1))was prepared by reacting the “T template” oligonucleotide (see below)with para-formyl benzoic acid N-hydroxysuccinimidyl ester as describedpreviously (Gartner et al. (2002) ANGEW. CHEM. INT. ED. 41: 1796; (2002)ANGEW. CHEM. 114:1874). Template 1 was combined with reagents 2 and 3(WO 2004/016767 A2) in aqueous 200 mMN-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES) bufferat pH 8.5 with 1 M NaCl, (63 nM template and 125 nM of each reagent).Reactions proceeded for up to 1 hour at 25° C.

The results of denaturing polyacrylamide gel electrophoresis analysis ofthese reactions is shown in (WO 2004/016767 A2, FIG. 38B). The 30-base Tarchitecture template (WO 2004/016767 A2, (1)) containing an aldehydegroup was present in lanes 1-2 and lanes 5-10. A template lacking thealdehyde group but otherwise identical to (1) was present in lanes 3 and4. DNA-linked phosphine reagent (WO 2004/016767 A2, (2)) was present inlanes 3-6 and lanes 9-10. DNA-linked alpha-iodoamide reagent (WO2004/016767 A2, (3)) was present in lanes 3-4 and lanes 7-10. Lanes 1,3, 5, 7 and 9 show reactions after 30 minutes. Lanes 2, 4, 6, 8, and 10show reactions after 1 hour.

T Architecture-mediated Conversion of Compound 5 to 8.

The 5′-propargylglycine linked oligonucleotide (WO 2004/016767 A2, (6))was generated by combining the corresponding T (n=−1) 5′-amine-linkedreagent oligonucleotide (see below) with 2 mg/mLbis(sulfosuccinimidyl)suberate in 9:1 200 mM sodium phosphate pH 7.2:DMFfor 10 minutes at 25° C., followed by treatment with 0.3 vol of 300 mMracemic propargylglycine in 300 mM NaOH for 2 hours at 25° C. The3′-azido linked oligonucleotide (WO 2004/016767 A2, (7)) was generatedby combining the T (n=1) amine-linked reagent oligonucleotide (seebelow) with 2 mg/mL (N-15 hydroxysuccinimidyl)-4-azidobenzoate in 9:1200 mM sodium phosphate pH 7.2:DMF for, 2 hours at 25° C. Reagents 6 and7 (WO 2004/016767 A2) were purified by gel filtration and reverse-phaseHPLC. Template 5 and reagents 6 and 7 were combined in aqueous 100 mMMOPS pH 7.0 in the presence of 1 M NaCl and 20 mM DMT-MM for 12 hours(60 nM template, 120 nM reagents) at 25° C. Copper (II) sulfatepentahydrate and sodium ascorbate were then added to 500 uM each. After1 hour at 25° C., reactions were quenched by ethanol precipitation.

DNA Oligonucleotide Sequences Used. E or Omega template: 5′-H₂N-GGTACGAATTCG ACT CGG GAA TAC CAC CTT [SEQ ID NO: 58]. H template:5′-H₂N-CGC GAG CGT ACG CTC GCG GGT ACG AAT TCG ACT CGG GAA TAC CAC CTT[SEQ ID NO: 59]. T template: 5′-GGT ACG AAT TCG AC(dT-NH₂) CGG GAA TACCAC CTT [SEQ ID NO: 60]. E or H reagent (n=1): 5′-AAT TCG TAC C—NH₂ [SEQID NO: 61]. E or H reagent (n=10): 5′-TCC CGA GTC G-NH₂ [SEQ ID NO: 62].E or H reagent (n=20): 5′-AAG GTG GTA T-NH₂ [SEQ ID NO: 63]. MismatchedE or H reagent: 5′-TCC CTG ATC G-NH₂ [SEQ ID NO: 64]. Omega-3 reagent(ra=10): 5′-TCC CGA GTC GAC C—NH₂ [SEQ ID NO: 65]. Omega-4 reagent(ra=10): 5′-TCC CGA GTC GTA CC—NH₂ [SEQ ID NO: 66]. Omega-5 reagent(n=10): 5′-TCC CGA GTC GGT ACC—NH₂-[SEQ ID NO: 67]. Omega-3 reagent(n=20): 5′-AAG GTG GTA TAC C—NH₂ [SEQ ID NO: 68]. Omega-4 reagent(n=20): 5′-AAG GTG GTA TTA CC—NH₂ [SEQ ID NO: 69]. Omega-5 reagent(n=20): 5′-AAG GTG GTA TGT ACC—NH₂ [SEQ ID NO: 70]. Mismatched Omega-3reagent: 5′-TCC CTG ATC GAC C—NH₂ [SEQ ID NO: 71]. Mismatched Omega-4reagent: 5′-TCC CTG ATC GTA CC—NH₂ [SEQ ID NO: 72].

Mismatched Omega-5 reagent: 5′-TCC CTG ATC GGT ACC NH₂ [SEQ ID NO: 73].T reagent (n=I): 5′-GGT′5 ATT CCC G-NH₂ [SEQ ID NO: 74]. T reagent(n=2): 5′-TGG TAT TCC C—NH₂ [SEQ ID NO: 75]. T reagent (n=3): 5′-GTGGTA′TTC C—NH₂ [SEQ ID NO: 76]. T reagent, \n=4): 5′-GGT GGT ATT C—NH₂[SEQ ID NO: 77]. T reagent (n=5): 5′-AGG TGG TAT T-NH₂ [SEQ ID NO: 78].T reagent (n=−1): 5′—NH₂-GTC GAA TTC G [SEQ ID NO: 79], T reagent (n=−4)for 2: 5′-[C₁₂-amine linker]-AAT TCG TAG C [SEQ ID NO: 80]. Reactionyields were quantitated by denaturing polyacrylamide gel electrophoresisfollowed by ethidium bromide staining, UV visualization, and CCD-baseddensitometry of product and template starting material bands. Yieldcalculations assumed that templates and products were denatured and,therefore, stained with comparable intensity per base; for those casesin which products are partially double-stranded during quantitation,changes in staining intensity may result in higher apparent yields.Representative reaction products were characterized by MALDI massspectrometry in addition to denaturing polyacrylamide gelelectrophoresis.

Melting curves were obtained on a Hewlett-Packard 8453 UV-visiblespectrophotometer using a Hewlett-Packard 89090A Peltierthermocontroller. Absorbances of template-reagent pairs (1.5 uM each) at260 nm were measured every 1° C. from 20° C. to 80° C. holding for 1minute at each temperature in either phosphate-buffered saline (“PBS,”137 mM NaCl, 2.7 mM potassium chloride, 1.4 mM potassium phosphate, 10mM sodium phosphate, pH 7.4) or in high salt phosphate buffer (“HSB,” 50mM sodium phosphate pH 7.2, 1 M NaCl).

Example 18 Functionalisation of Oligonucleotides

This is an example of how oligonucleotides may be functionalized fortheir further manipulation in stage 1 or stage 2 synthesis schemes. Italso describes a stage 1 amine acylation reaction. The description ofthe experiment is taken from (Liu et al., WO 2004/016767 A2, p. 131).The figures referred to are from the same patent application.

2-bromopropionamide-NNS Esters.

200 mg JV-hydroxysuccinimide (Pierce, Rockford, Ill., USA) was dissolvedin anhydrous CH₂Cl₂ together with 1.1 equivalents of 2-bromopropionicadd (either racemic, (R)-, or (5)-) and 2 equivalents of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) (Aldrich). The2-bromopropionic acid enantiomers were >95% enantiopure as judged bychiral HPLC (5% isopropanol in hexanes, (R,R) WHELK 01 chiral phase,detection at 220 nm). The reaction was maintained at room temperatureand complete after 1.5 hours as judged by TLC (EtOAc). The crudereaction mixture was extracted with 2.5% sodium hydrogen sulfate (NaHSO₄to remove the excess EDC. The organic phase was washed with brine, driedover magnesium sulfate (MgSO₄, and concentrated in vacuo. The residuewas dried and used directly for DNA functionalization.

5′-Functionalization of Oligonucleotides.

An NHS ester prepared as described above was dissolved in DMSO. Up to150 ug of a 5′-amino DNA oligonucleotide was combined with 3 mg/mL NHSester (final reaction=10% DMSO) in 200 mM sodium phosphate (pH=7.2) atroom temperature for 2 hours. The functionalized oligonucleotides werepurified by gel filtration and reverse-phase HPLC, and werecharacterized by denaturing PAGE and MALDI-TOF mass spectrometry.

3′-thiol Modified Oligonucleotides.

The 3′ thiol group was incorporated by standard automated DNA synthesisusing 3′-disulfide-linked CPG (Glen Research, Sterling, 20 Virginia,USA). Following oligonucleotide synthesis, the disulfide was cleavedwith 50 mM DTT, 1M TAPS (pH=8.0) at room temperature for 1 hour andpurified by gel filtration before being used in DNA-templated reactions.

Example 19 One-Pot Simultaneous Stage 2 Synthesis Involving AmineConjugate Addition, Thiol Conjugate Addition, Nitro-Michael Addition,Reductive Amination, Amine Acylation, And Wittig Olefination

This is an example of a number of templated reactions that are executedsimultaneously in one solution, giving high yields of all reaction typestested. The same reaction conditions may be applied to stage 1synthesis, except that the molecule fragments must be added in hg herconcentrations (preferably 10-100 mM). The description of theexperiments is taken from (Liu et al., WO 2004/016767 A2, example 7, p.137-142). The figures referred to are from the same patent application.

This example demonstrates that oligonucleotides can simultaneouslydirect several different synthetic reaction types within the samesolution, even though the reactants involved would be cross-reactiveand, therefore, incompatible under traditional synthesis conditions.These findings also demonstrate that it is possible to perform a one-potdiversification of synthetic library precursors into products usingmultiple, simultaneous and not necessarily compatible reaction types.

The ability of DNA templates to mediate diversification using differentreaction types without spatial separation was initially tested bypreparing three oligonucleotide templates of different DNA sequences(1a-3a) (WO 2004/016767 A2) functionalized at their 5′ ends withmaleimide groups and three oligonucleotide reagents (4a-6a) (WO2004/016767 A2) functionalized at their 3′ ends with an amine, thiol, ornitroalkane group, respectively (WO 2004/016767 A2, FIG. 46). The DNAsequences of the three reagents each contained a different 10-baseannealing region that was complementary to ten bases, near the 5′ end ofeach of the templates. Combining 1a with 4a, 2a with 5a, or 3a with 6ain three separate vessels at pH 8.0 resulted in the expectedDNA-templated amine conjugate addition, thiol conjugate addition, ornitro-Michael addition products 7-9 (WO 2004/016767 A2, FIG. 46, lanes1-3).

To distinguish the nine possible reaction products that could begenerated upon combining 1a-6a, the lengths of template oligonucleotideswere varied to include 11, 17, or 23 bases and the lengths of reagentoligonucleotides were varied to include 14, 16, or 18 bases. Differencesin oligonucleotide length were achieved using extensions distal from thereactive groups that did not significantly affect the efficiency ofDNA-templated reactions. This design permitted all nine possiblereaction products (linked to 25, 27, 29, 31, 33, 35, 37, 39, or 41 basesof DNA) to be distinguished by denaturing polyacrylamide gelelectrophoresis.

A solution containing all three templates (1a-3a) was combined with asolution containing all three reagents (4a-6a) at pH 8.0. The resultingreaction exclusively generated the three desired products 7, 8, and 9 oflengths 25, 33, and 41 bases indicating that only the three reactionscorresponding to the complementary template-reagent pairs took place (WO2004/016767 A2, FIG. 46, lane 4). Formation of the other six possiblereaction products was not detected by densitometry (<5% reaction). Incontrast, individually reacting templates and reagents containing thesame, rather than different, 10-base annealing regions permitted theformation of all possible products (WO 2004/016767 A2, FIG. 46, lane 5).This result demonstrates the ability of DNA-templated synthesis todirect the selective one-pot transformation of a single functional groupinto three distinct types of products (in this example, maleimide intosecondary amine, thioether, or a-branched nitroalkane).

To test the ability of this diversification mode to support one-potreactions requiring non-DNA-linked accessory reagents, an analogousexperiment was conducted with two aldehyde-linked reagents either 14 or16 bases in length (WO 2004/016767 A2, (4b) or (5b), respectively) and acomplementary 11-base amine-linked template (WO 2004/016767 A2, (1b)) ora 17-base phosphorane-linked template (WO 2004/016767 A2, (2b)).Combining 1b and 4b at pH 8.0 in the presence of 3 mM NaBH₃CN resultedin the DNA-templated reductive animation product 10 (WO 2004/016767 A2),while 2b and 5b under the same conditions generated Wittig olefinationproduct 11 (WO 2004/016767 A2, FIG. 46). Mixing all four reactantstogether in one pot resulted in an identical product distribution as thecombined individual Wittig olefination or reductive animation reactions(WO 2004/016767 A2, FIG. 46). No reaction between amine 1b and aldehyde5b or between phosphorane 2b and aldehyde 4b was detected (WO2004/016767 A2, FIG. 46, lane 8 versus lane 9).

The generality of this approach was explored by including multiplereaction types that required different accessory reagents. Threeamine-linked templates (1c-3c) (WO 2004/016767 A2) of length 11, 17, or23 bases were combined with an aldehyde-, carboxylic acid-, ormaleimide-linked reagent (4c-6c) (WO 2004/016767 A2) 14, 16, or 18 basesin length, respectively, at pH 8.0 in the presence of 3 mM NaBH₃CN, 10mM 1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimide (EDC), and 7.5 mMN-hydroxylsulfosuccinimide

(sulfo-NHS). The reactions containing all six reactants afforded thesame three reductive animation, amine acylation, or conjugate additionproducts (12-14) (WO 2004/016767 A2) that were generated from theindividual reactions containing one template and one reagent and did notproduce detectable quantities of the six possible undesired productsarising from non-DNA-templated reactions (WO 2004/016767 A2, FIG. 46,lanes 10-14). Collectively, these results indicate that DNA-templatedsynthesis can direct simultaneous reactions between several mutuallycross-reactive groups in a single pot to yield only thesequence-programmed subset of many possible products.

The above three examples each diversified a single functional group(maleimide, aldehyde, or amine) into products of different reactiontypes. A more general format for the one-pot diversification of aDNA-templated synthetic library into products of multiple reaction typeswould involve the simultaneous reaction of different functional groupslinked to both reagents and templates. To examine this possibility, sixDNA-linked nucleophile templates (15-20) (WO 2004/016767 A2) and sixDNA-linked electrophile reagents (21-25) (WO 2004/016767 A2)collectively encompassing all of the functional groups used in the abovethree examples (amine, aldehyde, maleimide, carboxylic acid,nitroalkane, phosphorane, and thiol) were prepared (WO 2004/016767 A2,FIG. 47). These twelve DNA-linked reactants could, in theory, undergosimultaneous amine conjugate addition, thiol, conjugate addition,nitro-Michael addition, reductive amination, amine acylation, and Wittigolefination in the same pot, although the apparent second order rateconstants of these six reactions vary by more than 10-fold.

Determining the outcome of combining all twelve reagents and templatesin a single pot by using oligonucleotides of varying lengths; isdifficult due the large number (at least 28) of possible products thatcould be generated. Accordingly, the length of the reagents as 15, 20,25, 30, 35, or 40 bases were varied but the length of the templates wasfixed at 11 bases (WO 2004/016767 A2, FIG. 47). Each of the sixcomplementary template-reagent pairs when reacted separately at pH 8.0in the presence of 3 mM NaBH₃CN; 10 mM EDC, and 7.5 mM sulfo-NHSgenerated the expected amine conjugate addition, thiol conjugateaddition, nitro-Michael addition, reductive amination, amine acylation,or Wittig olefination products (WO 2004/016767 A2, FIG. 47). Reactionefficiencies were greater than 50% relative to the correspondingindividual reactions despite having to compromise between differingoptimal reaction conditions. Templates 15-20 (WO 2004/016767 A2) werealso prepared in a 3′-biotinylated form. The biotinylated templatesdemonstrated reactivities indistinguishable from those of theirnon-biotinylated counterparts (WO 2004/016767 A2, FIG. 47).

Six separate reactions each containing twelve reactants then wereperformed at pH 8.0 in the presence of 3 mM NaBH₃CN, 10 mM EDC, and 7.5mM sulfo-NHS (WO 2004/016767 A2, FIG. 48). Each reaction contained adifferent biotinylated template (15, 16, 17, 18, 19, or 20) togetherwith five non-biotinylated templates (from 15-20) (WO 2004/016767 A2)and six reagents (21-25) (WO 2004/016767 A2). These reactions wereinitiated by combining a solution containing 15-20 with a solutioncontaining 21-25. The products that arose from each biotinylatedtemplate were captured with streptavidin-coated magnetic beads andidentified by denaturing gel electrophoresis. Because the six reagentsin each reaction contained oligonucleotides of unique lengths, theformation of any reaction products involving the biotinylated templatesand any of the reagents could be detected. In all six cases, thebiotinylated template formed only the single product programmed by itsDNA sequence (WO 2004/016767 A2, FIG. 48) despite the possibility offorming up to five other products in each reaction. Taken together,these findings indicate that reactions of significantly different ratesrequiring a variety of non-DNA-linked accessory reagents can be directedby DNA-templated synthesis in the same solution, even when bothtemplates and reagents contain several different cross-reactivefunctional groups. The ability of DNA templates to direct multiplereactions at concentrations that exclude non-templated reactions fromproceeding at appreciable rates mimics, in a single solution, aspatially separated set of reactions.

Compared to the use of traditional synthetic methods, generatinglibraries of small molecules by DNA-templated synthesis is limited byseveral factors including the need to prepare DNA-linked reagents, therestriction of aqueous, DNA-compatible chemistries, and the reliance oncharacterization methods such as mass spectrometry and electrophoresisthat are appropriate for molecular biology-scale (pg to ug) reactions.On the other hand, DNA-templated synthesis (i) allows the direct invitro selection (as opposed to screening) and amplification of syntheticmolecules with desired properties, (ii) permits the preparation ofsynthetic libraries of unprecedented diversity, and (iii) requires onlyminute quantities of material for selection and identification of activelibrary members. In addition, this example demonstrates that potentiallyuseful modes of reactivity not possible using current synthetic methodscan be achieved in a DNA-templated format. For example, six differenttypes of reactions can be performed simultaneously in one solution,provided that required non-DNA-linked accessory reagents are compatible.This reaction mode permits the diversification of synthetic smallmolecule libraries using different reaction types in a single solution.

Materials and Methods Synthesis of Templates and Reagents

Oligonucleotides were synthesized using standard automated solid-phasetechniques. Modified phosphoramidites and controlled-pore glass supportswere obtained from Glen Research, Sterling, Va., USA. Unless otherwisenoted, functionalized templates and reagents were synthesized byreacting 5′-H₂N(CH₂O)₂ terminated oligonucleotides (for templates) or3′-OPO₃—CH₂CH(CH₂OH)(CH₂)₄NH₂ terminated oligonucleotides (for reagents)in a 9:1 mixture of aqueous 200 mM pH 7.2 sodium phosphate buffer: DMFcontaining 2 mg/mL of the appropriate N-hydroxysuccinimide ester(Pierce, Rockford, Ill., USA) at 25° C.

For the aldehyde and nitroalkane-linked oligonucleotides (4b, 4c, 5b,6a, 17, 24, and 26, FIGS. 46 and 47, WO 2004/016767 A2) the NHS esterswere generated by combining the appropriate carboxylic acid (900 mM inDMF) with equal volumes of dicyclohexylcarbodiimide (900 mM in DMF) andNHS (900 mM in DMF) for 90 minutes. Phosphorane-linked oligonucleotides(2b and 20, FIGS. 46 and 47, WO 2004/016767 A2) were prepared by a 90minute reaction of the appropriate amino-terminated oligonucleotide with0.1 volumes of a 20 mg/mL DMF solution of the NHS ester of iodoaceticacid (SIA, Pierce, Rockford, Ill., USA) in pH 7.2 buffer as above,followed by addition of 0.1 volumes of a 20 mg/mL solution of4-diphenylphosphinobenzoic acid in DMF.

Thiol-linked template 16 was synthesized by reacting ethylene glycolbis(succinimidyisuccinate) (EGS, Pierce, Rockford, Ill., USA) with theappropriate oligonucleotide for 15 minutes, followed by addition of 0.1volumes of 300 mM 2-aminoethanethiol. Reagent 5a was synthesized using3′-OP0₃—(CH₂)₃5S(CH₂)₃ODMT functionalized controlled-pore glass (CPG)support and reduced prior to use according to the manufacturer'sprotocol.

The 3′-biotinylated oligonucleotides were prepared using biotin-TEG¹ CPG(Glen Research, Sterling, Va., USA). Products arising from biotinylatedtemplates were purified by mixing with 1.05 equivalents ofstreptavidin-linked magnetic beads (Roche), washing twice with 4 Mguanidinium hydrochloride, and eluting with aqueous 10 mM Tris pH 7.6with 1 mM biotin at 80° C.

Synthesis of Linkers

Linkers between DNA oligonucleotides and the functional groups in 1a-6care as follows. 1b and 1c: DNA-5′—NH₂; 1a, 2a-2c,3a, and 3c:DNA-5′—O(CH₂)₂—O—(CH₂)₂—NH—; 5a: DNA-3′-O-(CH₂)₃SH; 4a-4-c, 5b, 5c, 6a,and 6c: DNA-3′-O—CH₂CH(CH₂OH)(CH₂)₄NH—. Oligonucleotide sequences usedto generate all possible products in (WO 2004/016767 A2, FIG. 46, lanes5, 9, and 14), with annealing regions underlined: R-TATCTACAGAG-3′ [SEQID NO: 106] (1a-1c); R-TATCTACAGAGTAGTCT-3′ [SEQ ID NO: 107] (2a-2c);R-TATCTACAGAGTAGTCTAATGAC-3′ [SEQ ID NO: 108] (3a-3c);5′-CAGCCTCTGTAGAT-R [SEQ ID NO: 109] (4a-4-c); T-CTCAGCCTCTGTAGAT-R [SEQID NO: 110] (5a-5c); 5′-GGCTCAGCCTCTGTAGAT-R [SEQ ID NO: 111] (6a-6c).Functionalized templates and reagents were purified by gel filtration(Sephadex G-25) followed by reverse-phase HPLC (0.1 M triethylammoniumacetate/acetonitrile gradient). Representative functionalized templatesand reagents were further characterized by MALDI mass spectrometry.

Reaction Conditions

All reactions were performed by dissolving reagents and templates inseparate vessels in pure water before combining them into a solution of50 mM aqueous TAPS buffer, pH 8.0, 250 mM NaCl at 25° C. for 16 hourswith DNA-linked reactants at 60 nM (WO 2004/016767 A2, FIG. 47) or at12.5 nM (WO 2004/016767 A2, FIGS. 47 and 48). NaBH₃CN, EDC, andsulfo-NHS were present when appropriate as described. Products wereanalyzed by denaturing polyacrylamide gel electrophoresis using ethidiumbromide staining and UV transillumination: Differences in charge states,attached functional groups, and partial secondary structure resulted inmodest variations in gel mobility for different functionalizedoligonucleotides of the same length (FIGS. 46-48).

Example 20 Selection for Bifunctional Molecules Capable of Binding to aMacromolecular Target

This is an example of a selection against 6 protein targets, by affinityselection on immobilized protein (subprocess i). The experiments aredescribed in detail in the patent application (Liu et al., WO2004/016767 A2, example 11, p. 171-182).

Six proteins, GST, Carbonic anhydrase, Papain, Trypsin, Chymotrypsin,and Strepavidin, were immobilized on NHS-activated Sepharose 4 fast flowbeads. For each of the proteins, a known ligand was prepared and linkedto a unique DNA sequence. Solutions containing DNA-linked proteinligands and DNA-linked negative controls were used to simulate librariesof bifunctional molecules. The selections were performed by firstincubating the DNA-linked ligands with immobilized protein, then beadswere washed, and finally the DNA of the DNA-linked ligands that bound tothe beads was amplified by PCR, to reveal the efficiency of the modelselection experiment. All proteins were enriched more than 50-fold.

Example 21 Iterated Selection on Immobilized Target (Subprocess viii)

This is an example of iterated rounds of selection and elution withoutintervening amplification of the bifunctional molecule (subprocess viii,above). The description of the experiments is taken from (Liu et al., WO2004/016767 A2, example 11, p. 173). The figures referred to are fromthe same patent application.

Selections can be iterated to multiply the net enrichment of desiredmolecules. To test this possibility with DNA-lirjked syntheticmolecules, a 1:1,000 mixture of DNA-linked phenyl sulfonamide(3):DNA-linked N-formyl-Met-Leu-Phe (2) (WO 2004/016767 A2) wassubjected to a selection for binding carbonic anhydrase. The moleculessurviving the first selection were eluted and directly subjected to asecond selection using fresh immobilized carbonic anhydrase. PCRamplification and restriction digestion revealed that the first round ofselection yielded a 1:3 ratio of (3):(2), representing a 3,30-foldenrichment for the DNA-linked phenyl sulfonamide. The second round ofselection further enriched (3) by more than 30-fold, such that the ratioof (3):(2) following two rounds of selection exceeded 10:1 (>10⁴-foldnet enrichment). Similarly, three rounds of iterated selection were usedto enrich a 1:10⁶ starting ratio of (3):DNA-linked biotin (4) by afactor of 5×10⁶ into a solution containing predominantly DNA-linkedphenyl sulfonamide (3) (see WO 2004/016767 A2, FIG. 81). These findingsdemonstate that enormous net enrichments for DNA-linked syntheticmolecules can be achieved through iterated selection, and suggest thatdesired molecules represented as rarely as 1 part in 10⁶ withinDNA-templated synthetic libraries may be efficiently isolated in thismanner.

Example 22 Stage 2 Reactions Reductive Amination, Amine Acylation,Carbon-Carbon Forming Reactions, and Organometallic Coupling Reactions

This is an example of reactions that can be employed in a stage 2synthesis. By maintaining a high concentration of molecule fragments(e.g. 10-100 mM), the conditions applied to the templated synthesishereunder, can be applied to stage 1 synthesis as well, using the samereaction types. The description of the experiments is taken from (Liu etal., WO 2004/016767 A2, example 2, p. 107-112). The figures referred toare from the same patent application.

As described in detail herein, a variety of chemical reactions forexample, DNA-templated organometallic couplings and carbon-carbon bondforming reactions can be utilized to construct small molecules.

The ability of DNA-templated synthesis to direct reactions that requirea non-DNA-linked activator, catalyst or other reagent in addition to theprincipal reactants has also been demonstrated herein. To test theability of DNA-templated synthesis to mediate such reactions withoutrequiring structural mimicry of the DNA-templated backbone,DNA-templated reductive animations between an amine-linked template (1)(WO 2004/016767 A2) and benzaldehyde- or glyoxal-linked reagents (3) (WO2004/016767 A2) with millimolar concentrations of sodiumcyanoborohydride (NaBH₃CN) at room temperature in aqueous solutions canbe performed (see WO 2004/016767 A2, FIG. 23A). Significantly, productsformed efficiently when the template and reagent sequences werecomplementary, while control reactions in which the sequence of thereagent did not complement that of the template, or in which NaBH₃CN wasomitted, yielded no significant product (see WO 2004/016767 A2, FIGS.23A-23D and 24). Although DNA-templated reductive aminations to generateproducts closely mimicking the structure of double-stranded DNA havebeen previously reported (see, for example, Li et al. (2002) J. AM.CHEM. SOC. 124: 746 and Gat et al. (1998) BIOPOLYMERS 48:19), theseresults demonstrate that reductive animation to generate structuresunrelated to the phosphoribose backbone can take place efficiently andsequence-specifically.

Referring to (WO 2004/016767 A2, FIGS. 25A-25B, DNA-templated amide bondformations between amine-linked templates 4 and 5 and carboxylate-linkedreagents 6-9 mediated by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide(EDC) and N-hydroxylsulfosuccinimide (sulfo-NHS) generated amideproducts in good yields at pH 6.0, 25° C. Product formation was (i)sequence-specific, (ii) dependent on the presence of EDC, and (iii)insensitive to the steric encumbrance of the amine or carboxylate.Efficient DNA-templated amide formation was also mediated by thewater-stable activator4-(4,6-dimethoxy-1,3,5-trizin-2-yl)-4-methylmorpholinium chloride(DMT-MM) instead of EDC and sulfo-NHS (WO 2004/016767 A2, FIGS. 24 and25A-25B). The efficiency and generality of DNA-templated amide bondformation under these conditions, together with the large number ofcommercially available chiral amines and carboxylic acids, make thisreaction an attractive candidate in future DNA-templated syntheses ofstructurally diverse small molecule libraries.

Carbon-carbon bond forming reactions are also important in both chemicaland biological syntheses and thus several such reactions can be utilizedin a nucleic acid-templated format. Both the reaction ofnitroalkane-linked reagent (10) (WO 2004/016767 A2) with aldehyde-linkedtemplate (11) (WO 2004/016767 A2) (nitro-aldol or Henry reaction) andthe conjugate addition of 10 to maleimide-linked template (12) (WO2004/016767 A2) (nitro-Michael addition) proceeded efficiently and withhigh sequence specificity at pH 7.5-8.5, 25° C. (WO 2004/016767 A2,FIGS. 23A and 24). In addition, the sequence-specific DNA-templatedWittig reaction between stabilized phosphorus ylide reagent 13 (WO2004/016767 A2) and aldehyde-linked templates 14 or 11 (WO 2004/016767A2) provided the corresponding olefin products in excellent yields at pH6.0-8.0, 25° C. (WO 2004/016767 A2, FIGS. 23B and 24). Similarly, theDNA templated 1,3-dipolar cycloaddition between nitrone-linked reagents15 and 16 (WO 2004/016767 A2) and olefin-linked templates 12, 17 or 18also afforded products sequence specifically at pH 7.5, 25° C. (WO2004/016767 A2, FIGS. 23B, 23C and 24).

In addition to the reactions described above, organometallic couplingreactions can also be utilized in the present invention. For example,DNA-templated Heck reactions were performed in the presence ofwater-soluble Pd precatalysts. In the presence of 170 mM Na₂PdCl₄, aryliodide-linked reagent 19 (WO 2004/016767 A2) and a variety ofolefin-linked templates including maleimide 12, acrylamide 17, vinylsulfone 18 or cinnamamide 20 (WO 2004/016767 A2) yielded Heck couplingproducts in modest yields at pH 5.0, 25° C. (WO 2004/016767 A2, FIGS.23D and 24). For couplings with olefins 17, 18 and 20, adding twoequivalents of P(p-SO₃C₆H₄)₃ per equivalent of Pd prior to template andreagent addition typically increased overall yields by 2-fold.'. Controlreactions containing sequence mismatches or lacking Pd precatalystyielded no product.

In order to evaluate the ability of the DNA-templated reactions to takeplace efficiently when reactants are separated by distances relevant tolibrary encoding, the yields of reductive animation, amide formation,nitro-aldol addition, nitro-Michael addition, Wittig olefination,dipolar cycloaddition, and Heck coupling reactions were compared wheneither zero {n˜0) or ten (n=10) bases separated the annealed reactivegroups. Among the reactions described here, amide bond formation,nitro-aldol addition, Wittig olefination, Heck coupling, conjugateaddition of thiols to maleimides and S_(N)2 reaction between thiols andalpha-iodo amides demonstrate comparable product formation when reactivegroups are separated by zero or ten bases (WO 2004/016767 A2, FIG. 26B).FIG. 26B shows the results of denaturing polyacrylamide gelelectrophoresis of a DNA-templated Wittig olefination betweencomplementary 11 and 13 with either zero bases (lanes 1-3) or ten bases(lanes 4-6) separating the annealed reactants. Product yields after 13hours at both distances were nearly quantitative.

Control reactions containing sequence mismatches yielded no detectableproduct. These findings indicate that these reactions can be encodedduring synthesis by nucleotides that are distal from the reactive end ofthe template without significantly impairing product formation.

In addition to the DNA-templated S_(N)2 reaction, conjugate addition,vinyl sulfone addition, amide bond formation, reductive animation,nitro-aldol (Henry reaction), nitro Michael, Wittig olefination,1,3-dipolar cycloaddition and Heck coupling reactions described above, avariety of additional reagents can also be utilized in the method of thepresent invention. For example, as depicted in (WO 2004/016767 A2, FIG.27), powerful aqueous DNA-templated synthetic reactions including, butnot limited to, the Lewis acid-catalysed aldol addition, Mannichreaction, Robinson annulation reactions, additions of allyl indium, zincand tin to ketones and aldehydes, Pd-assisted allylic substitution,Diels-Alder cycloadditions, and hetero-Diels-Alder reactions can beutilized efficiently in aqueous solvent and are importantcomplexity-building reactions.

Taken together, these results expand considerably the reaction scope ofDNA-templated synthesis. A wide variety of reactions can proceedefficiently and selectively when the corresponding reactants areprogrammed with complementary sequences. By augmenting the repertoire ofknown DNA-templated reactions to include carbon-carbon bond forming andorganometallic reactions (nitro-aldol additions, nitro-Michaeladditions, Wittig olefinations, dipolar cycloadditions, and Heckcouplings, in addition to previously reported amide bond formation (see,Schmidt et al (1997) NUCLEIC ACIDS RES. 25:4792; Bruick et al. (1996)CHEM. BIOL. 3: 49), imine formation (Czlapinski: ̂ al. (2001) J. AM.CHEM. SOC. 123: 8618), reductive amination (Lie/ al. (2002) J. AM. CHEM.SOC. 124: 746; Gat et al. (1998) BiOPOLYMERS 48:19), S_(N)2 reactions(Gartner et al. (2001) J. AM. CHEM. SOC. 123: 6961; Xu et al. (2001)NAT. BIOTECHNOL. 19: 148; Herrlein et al. (1995) J. AM. CHEM. SOC. 117:10151), conjugate addition of thiols (Gartner et al. (2001) J. AM. CHEM.SOC. 123: 6961), and phosphoester or phosphonamide formation (Orgel etal. (1995) Ace. CHEM. RES. 28: 109; Luther et al. (1998) NATURE 396:245), these results may permit the sequence-specific translation oflibraries of DNA into libraries of structurally and functionally diversesynthetic products.

Because minute quantities of templates encoding desired molecules can beamplified by PCR, the yields of DNA-templated reactions arguably areless critical than the yields of traditional synthetic transformations.Nevertheless, many of the reactions discussed in this example proceedefficiently.

Materials and Methods

Functionalized templates and reagents were typically prepared byreacting 5′—NH₂ terminated oligonucleotides (for template 1),5′—NH₂—(CH₂O)₂ terminated oligonucleotides (for all other templates) or3′-OP0₃—CH₂CH(CH₂OH)(CH₂)₄NH₂ terminated nucleotides (for all reagents)with the appropriate NHS esters (0.1 volumes of a 20 mg/mL solution inDMF) in 0.2 M sodium phosphate buffer, pH 7.2, 25° C., for 1 hour toprovide the template and reagent structures shown in (WO 2004/016767 A2,FIGS. 23A-23D and 25A-25B). For amino acid linked reagents 6-9,3′-OPO₃CH₂CH(CH₂OH)(CH₂)₄NH₂ terminated oligonucleotides in 0.2 M sodiumphosphate-buffer, pH 7.2 were reacted with 0.1 volumes of a 100 mMbis[2-(succinimidyloxycarbonyloxy)ethyl]sulfone (BSOCOES, Pierce,Rockford, Ill., USA) solution in DMF for 10 minutes at 25° C., followedby 0.3 volumes of a 300 mM amino acid in 300 mM sodium hydroxide (NaOH)for 30 minutes at 25° C.

Functionalized templates and reagents were purified by gel filtrationusing Sephadex G-25 followed by reverse-phase HPLC (0.1 triethylammoniumacetate-acetonitrile gradient) and characterized by MALDI massspectrometry. For the DNA templated reactions described in (WO2004/016767 A2, FIGS. 23A-23D) reactions were conducted at 25° C. withone equivalent each of template and reagent at 60 nM final concentrationunless otherwise specified. Conditions: (a) 3 mM NaBH₃CN, 0.1M/V-[2-morpholinoethane]sulfonic acid (MES) buffer pH 6.0, 0.5 M NaCl,1.5 hours; b) 0.1 M N-tris[hydroxymethyl]methyl-3-aminopropanesulfonicacid (TAPS) buffer pH 8.5, 300 mM NaCl, 12 hours; c) 0.1 M pH 8.0 TAPSbuffer, 1 M NaCl, 5° C., 1.5 hours; d) 50 mM MOPS buffer pH 7.5, 2.8 MNaCl, 22 hours; e) 120 nM 19, 1.4 mM Na₂PdCl₄, 0.5 M NaOAc buffer pH5.0, 18 hours; (f) Premix NaaPdCL} with two equivalents of P(p-SO₃CeH₄)₃in water for 15 minutes, then add to reactants in 0.5 M NaOAc buffer pH5.0, 75 mM NaCl, 2 hours (final [Pc]=0.3 mM, [19]=120 nM). The olefingeometry of products from 13 and the regiochemistries of cycloadditionproducts from 14 and 16 are presumed but not verified (WO 2004/016767A2, FIGS. 23A-23D). Products were characterized by denaturingpolyacrylamide gel electrophoresis and MALDI mass spectrometry. For allreactions under the specified conditions, product yields of reactionswith matched template and reagent sequences were greater than 20-foldhigher than that of control reactions with scrambled reagent sequences.

The conditions for the reactions described in (WO 2004/016767 A2, FIGS.25A-25B) were: 60 nM template, 120 nM reagent, 50 mM DMT-MM in 0.1 MMOPS buffer pH 7.0, 1 M NaCl, for 16 hours at, 25° C.; or 60 nMtemplate, 120 nM reagent, 20 mM EDC, 15 mM sulfo-NHS, 0.1 M MES bufferpH 6.0, 1 M NaCl, for 16 hours at 25° C. In each row of the table in (WO2004/016767 A2, FIGS. 25A-258), yields of DMT-MM-mediated reactionsbetween reagents and templates complementary in sequence were followedby yields of EDC and sulfo-NHS-mediated reactions. In all cases, controlreactions with mismatched reagent sequences yielded little or nodetectable product and products were characterized by denaturingpolyacrylamide gel electrophoresis and MALDI mass spectrometry.

(WO 2004/016767 A2, FIG. 24) depicts the analysis by denaturingpolyacrylamide gel electrophoresis of representative DNA-templatedreactions listed in (WO 2004/016767 A2, FIGS. 23A-23D and 25A-25B). Thestructures of reagents and templates correspond to the numbering inFIGS. 23A-23D and 25A-25B. Lanes 1, 3, 5, 7, 9, 11: reaction of matched(complementary or “M”) reagents and templates under conditions listed inFIGS. 23A-23D and 25A-25B (the reaction between 4 and 6 was mediated.byDMT-MM). Lanes 2, 4, 6, 8, 10, 12: reaction of mismatched(non-complementary or “X”) reagents and templates under conditionsidentical to those in lanes 1, 3, 5, 7, 9 and 11, respectively.

The sequences of oligonucleotide templates and reagents are as follows(5′ to 3′ direction, n refers to the number of bases between reactivegroups when template and reagent are annealed as shown in (WO2004/016767 A2, FIG. 26A).

1: [SEQ ID NO: 45] TGGTACGAATTCGACTCGGG; 2 and 3 matched:[SEQ ID NO: 46] GAGTCGAATTCGTACC; 2 and 3 mismatched: [SEQ ID NO: 47]GGGCTCAGCTTCCCCA; 4 and 5: [SEQ ID NO: 48]GGTACGAATTCGACTCGGGAATACCACCTT; 6-9 matched (n = 10): [SEC ID NO: 49]TCCCGAGTCG; 6 matched (n = 0): [SEQ ID NO: 50] AATTCGTACC;6-9 mismatched: [SEQ ID NO: 51] TCACCTAGCA; 11,12,14,17,18, 20:[SEQ ID NO: 52] GGTACGAATTCGACTCGGGA; 10,13,16,19 matched:[SEQ ID NO: 53] TCCCGAGTCGAATTCGTACC; 10,13,16,19 mismatched:[SEQ ID NO: 54] GGGCTCAGCTTCCCCATAAT; 15 matched: [SEQ ID 20 NO: 55]AATTCGTACC; 15 mismatched: [SEQ ID NO: 56] TCGTATTCCA; template for n =10 vs. n = 0 comparison: [SEQ ID NO: 57] TAGCGATTACGGTACGAATTCGACTCGGGA.

Reaction yields were quantitated by denaturing PAGE followed by ethidiumbromide staining, UV visualization, and charge-coupled device(CCD)-based densitometry of product and template starting materialbands. Yield calculations assumed that templates and 25 products stainedwith equal intensity per base; for those cases in which products werepartially double-stranded during quantitation, changes in stainingintensity may have resulted in higher apparent yields.

Example 23 Different Stage 1 and Stage 2 Synthesis Schemes Employed in aGiven Series of Experiments

Because of the modular nature of the stage 1, stage 2 andselection/screening protocols, it is perfectly possible to generate afirst generation library using e.g. subprocess (1, i.e., no templatedsynthesis involved), then select (e.g. using subprocess i), and thenperform a second round of library generation, and this time use therecovered templates as templates, and therefore, perform a stage 2synthesis to make the enriched second generation library. Obviously, itis important to keep the same code for the same molecule fragments.

It may also be advantageous to select against immobilized target in thefirst round, and then in the second round perform in solution selectionexperiments for example, or some other selection experiment that sharefew of the same features as the first selection assay.

Example 24 Carrier Preparation by Several Different Routes

Because of the modular nature of the stage 1 synthesis procedures, thecarrier that are employed in a stage 2 synthesis can be prepared bydifferent synthetic routes. As an exmple, in order to make e.g. 2.000identifiers, with the ability to make 1.000.000 differenttemplate-encoded molecules, one could synthesize 625 carriers by twostep Lerner-like stage 1 synthesis (subprocess 1), using acylationreactions to link the molecule fragments; synthesize 1000 carriers usingthe DNA-routing approach by Harbury (subprocess 10), for exampleemploying reductive amination and nucleophilic aromatic substitutionreactions; synthesize 375 compounds by combinatorial chemistry andattach these to identifiers. Then use this pool of 2000 carriers in astage 2 synthesis to generate 1.000.000 bifunctional molecules.

Example 25 Stage 1 Synthesis Employing the Harbury and Halpin Method(Subprocess 10)

Subprocess 10 stage 1 synthesis involves a DNA sorting step, in whichthe identifiers to be linked to the molecule fragments are sortedaccording to their DNA sequences. Once the DNA has been sorted, themolecule fragments can be linked under conditions identical to theconditions described in the present invention, in particular, asdescribed in all of the above examples. Thus, the preferred reactions,reductive amination, Wittig reaction, acylation, alkylhalide alkylation,nucleophilic aromatic substitution, Heck coupling, cycloadditionreactions, sulfonoylation, isocyanide addition, Michael addition andothers, may be executed in exactly the same way as described here.

Applications of the present invention.

The methods of the present invention provide for the identification oforganic and inorganic molecules that are catalysts useful for thesynthesis of complex molecules from simple substrates, inorganiccompounds with useful properties as materials, may be used in thedegradation of plastics, animal feed processing, etc. Also, the methodscan be applied to identification of compounds with high affinity orselectivity for targets and surfaces, including protein targets, DNA,and other macromolecular structures, metal surfaces, plastics, etc. Suchcompounds may be useful as additives to paint, cement, textiles, andother substances where improved rigidity, strength, flexibility orstability is desired. New materials may be identified in this way,including superconductors and nanosensors.

Compounds that bind with high affinity and/or selectivity to protein,RNA, DNA, polysaccharides, or other molecules of an organism, may beused in diagnostics or as therapeutics.

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What is claimed is:
 1. A method for synthesizing an encoded moleculecomprising the steps of: a) Adding a linker molecule L to one or morereaction wells; b) Adding a molecule fragment to each of said reactionwells; c) Adding an oligonucleotide identifier to each of said reactionwells; d) Subjecting said wells to: conditions sufficient to allow saidmolecule fragments and said oligonucleotide identifiers to becomeattached to said linker molecule, or conditions sufficient for saidmolecule fragments to bind to other molecule fragments and sufficientfor said oligonucleotide identifiers to bind to other oligonucleotideidentifiers; e) Combining the contents of said one or more reactionwells; wherein at least one reactive group of the linker molecule Lreacts with a reactive group in the molecule fragment, or with areactive group in the oligonucleotide; wherein at least one reactivegroup of the molecule fragments reacts with a reactive group in thelinker molecule L, or with a reactive group in another moleculefragment, wherein at least one reactive group of the oligonucleotideidentifiers reacts with a reactive group in the linker L, or with areactive group in another oligonucleotide identifier; and wherein theoligonucleotide identifier added to each well in step c) identifies themolecule fragment added to the same well in step b).